Bioactive renal cells

ABSTRACT

The present invention concerns bioactive renal cell populations, renal cell constructs, and methods of making and using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/925,172, filed Mar. 19, 2018, which is continuation of U.S. patentapplication Ser. No. 13/697,206, which is a national stage application,filed under 35 U.S.C. § 371, of International Application No.PCT/US11/36347 filed May 12, 2011, which claims the benefit under 35U.S.C. § 119 of U.S. Provisional Application No. 61/473,111, filed Apr.7, 2011; U.S. Provisional Application No. 61/441,423, filed Feb. 10,2011; U.S. Provisional Application No. 61/413,382, filed Nov. 12, 2010;U.S. Provisional Application No. 61/412,933, filed Nov. 12, 2010; U.S.Provisional Application No. 61/388,765, filed Oct. 1, 2010; U.S.Provisional Application No. 61/376,586, filed Aug. 24, 2010; U.S.Provisional Application No. 61/372,077, filed Aug. 9, 2010; U.S.Provisional Application No. 61/371,888, filed Aug. 9, 2010; U.S.Provisional Application No. 61/353,895, filed Jun. 11, 2010; and U.S.Provisional Application No. 61/334,032, filed May 12, 2010, the entirecontents of each of which are hereby incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII text format and is hereby incorporatedby reference in its entirety. Said ASCII text copy, created on Mar. 19,2018, is named “050400_506C01US_Sequence_Listing.txt” and is 1,154 bytesin size.

FIELD OF THE INVENTION

The present invention relates to bioactive renal cell populations orfractions that lack cellular components as compared to a healthyindividual yet retain therapeutic properties, and methods of isolatingand culturing the same, as well as methods of treating a subject in needwith the cell populations. In addition, the present invention relates tomethods of providing regenerative effects to a native kidney usingbioactive renal cell populations.

BACKGROUND OF THE INVENTION

Chronic Kidney Disease (CKD) affects over 19 million people in theUnited States and is frequently a consequence of metabolic disordersinvolving obesity, diabetes, and hypertension. Examination of the datareveals that the rate of increase is due to the development of renalfailure secondary to hypertension and non-insulin dependent diabetesmellitus (NIDDM) (United States Renal Data System: Costs of CKD andESRD. ed. Bethesda, MD, National Institutes of Health, NationalInstitute of Diabetes and Digestive and Kidney Diseases, 2007 pp223-238)—two diseases that are also on the rise worldwide. Obesity,hypertension, and poor glycemic control have all been shown to beindependent risk factors for kidney damage, causing glomerular andtubular lesions and leading to proteinuria and othersystemically-detectable alterations in renal filtration function(Aboushwareb, et al., World J Urol, 26: 295-300, 2008; Amann, K. et al.,Nephrol Dial Transplant, 13: 1958-66, 1998). CKD patients in stages 1-3of progression are managed by lifestyle changes and pharmacologicalinterventions aimed at controlling the underlying disease state(s),while patients in stages 4-5 are managed by dialysis and a drug regimenthat typically includes anti-hypertensive agents, erythropoiesisstimulating agents (ESAs), iron and vitamin D supplementation.Regenerative medicine technologies may provide next-generationtherapeutic options for CKD. Presnell et al. WO/2010/056328 describeisolated renal cells, including tubular and erythropoietin(EPO)-producing kidney cell populations, and methods of isolating andculturing the same, as well as methods of treating a subject in needwith the cell populations. There is a need for new treatment paradigmsthat provide substantial and durable augmentation of kidney functions,to slow progression and improve quality of life in this patientpopulation.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for providing aregenerative effect to a native kidney. In one embodiment, the methodincludes the step of in vivo contacting the native kidney with productssecreted by an enriched renal cell population. In another embodiment,the products are secreted by an enriched renal cell population that isnot part of a construct, as described herein, e.g., the cell populationis not seeded on a scaffold. In one other embodiment, the products aresecreted from a renal cell construct comprising an enriched renal cellpopulation directly seeded on or in a scaffold. In another embodiment,the secretion of the products is bioresponsive to oxygen levels.Secretion may be induced by a less than atmospheric oxygen level. In oneother embodiment, the lower oxygen level is less than about 5% oxygen.

In one embodiment, the regenerative effect is a reduction inepithelial-mesenchymal transition (EMT). The reduction in EMT may beachieved via attenuation of TGF-β signalling and/or attenuation ofPlasminogen Activator Inhibitor-1 (PAI-1) signalling. In anotherembodiment, the regenerative effect is a reduction in renal fibrosisand/or a reduction in renal inflammation. In some embodiments, thereduction in inflammation may be mediated by NFκB. In one otherembodiment, the regenerative effect is characterized by differentialexpression of a stem cell marker in the native kidney. The expressionmay be an upregulation of marker expression in the in vivo contactednative kidney relative to expression in a non-contacted native kidney.

In one aspect, the enriched renal cell population includes one or morecell populations, i.e., an admixture, as described herein. In oneembodiment, the population includes a first cell population, B2, thatcontains an enriched population of tubular cells. In another embodiment,the population includes an admixture of human renal cells having a firstcell population, B2, and a second cell population, which contains one ormore of erythropoietin (EPO)-producing cells, glomerular cells andvascular cells. In one other embodiment, the second cell population is aB4 cell population. In yet another embodiment, the second cellpopulation is a B3 cell population.

In one embodiment, the admixture further includes a third cellpopulation having one or more of erythropoietin (EPO)-producing cells,glomerular cells and vascular cells. In another embodiment, the thirdcell population is a B4 cell population. In one other embodiment, thethird cell population is a B3 cell population.

In all embodiments, the B2 cell population has a density between about1.045 g/mL and about 1.052 g/mL. In all embodiments, the B4 cellpopulation has a density between about 1.063 g/mL and about 1.091 g/mL.In all embodiments, the B3 cell population has a density between about1.052 g/ml and about 1.063 g/ml.

In all embodiments, the enriched renal cell population may benon-autologous to the native kidney. In all embodiments, the enrichedrenal cell population may be autologous to the native kidney.

In all embodiments, the products include paracrine factors, endocrinefactors, juxtacrine factors, RNA, vesicles, microvesicles, exosomes, andany combination thereof. In one other embodiment, the vesicles includeone or more secreted products selected from the group consisting ofparacrine factors, endocrine factors, juxtacrine factors, and RNA. Inanother embodiment, the products are secreted from a renal cellconstruct comprising an enriched renal cell population directly seededon or in a scaffold.

In all embodiments, the scaffold may contain a biocompatible material.In all embodiments, the biocompatible material may be a hydrogel.

In one embodiment, the present invention provides methods of assessingwhether a kidney disease (KD) patient is responsive to treatment with atherapeutic. The method may include the step of determining or detectingthe amount of vesicles or their luminal contents in a test sampleobtained from a KD patient treated with the therapeutic, as compared toor relative to the amount of vesicles in a control sample, wherein ahigher or lower amount of vesicles or their luminal contents in the testsample as compared to the amount of vesicles or their luminal contentsin the control sample is indicative of the treated patient'sresponsiveness to treatment with the therapeutic. The vesicles may bekidney derived vesicles. The test sample may contain urine. The vesiclesmay contain a biomarker, which may be miRNA. The therapeutic may containan enriched population of renal cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show enrichment of epo-producing cell fraction fromfreshly-dissociated kidney tissue using a multi-layered step gradienttechnique (FIG. 1A—left panel) or a single-layer mixing gradienttechnique (FIG. 1B—right panel). Both methods result in the partialdepletion of non epo-producing cell components (predominantly tubularcells) from the epo band, which appears between 1.025 g/mL and 1.035g/mL.

FIG. 2 shows step gradients of “normoxic” (21% oxygen) and “hypoxic” (2%oxygen) rodent cultures that were harvested separately and appliedside-by-side to identical step gradients.

FIG. 3 shows step gradients of “normoxic” (21% oxygen) and “hypoxic” (2%oxygen) canine cultures that were harvested separately and appliedside-by-side to identical step gradients.

FIG. 4 shows histopathologic features of the HK17 and HK19 samples.

FIG. 5 shows high content analysis (HCA) of albumin transport in humanNKA cells defining regions of interest (ROI).

FIG. 6 shows quantitative comparison of albumin transport in NKA cellsderived from non-CKD and CKD kidney.

FIG. 7 depicts comparative analysis of marker expression betweentubular-enriched B2 and tubular cell-depleted B4 subfractions.

FIG. 8 depicts comparative functional analysis of albumin transportbetween tubular-enriched B2 and tubular cell-depleted B4 subfractions.

FIG. 9 shows expression of SOX2 mRNA in host tissue after treatment of5/6 NX rats.

FIG. 10 Western blot showing time course of expression of CD24, CD133,UTF1, SOX2, NODAL and LEFTY.

FIG. 11 depicts a time course of regenerative response index (RRI).

FIG. 12 provides a schematic for the preparation and analysis of UNFXconditioned media.

FIGS. 13A-13D show that conditioned media from UNFX cultures affectsmultiple cellular processes in vitro that are potentially associatedwith regenerative outcomes. FIG. 13A shows that UNFX-conditioned mediaattenuates TNF-a mediated activation of NF-kB. FIG. 13B shows thatUNFX-conditioned media increases proangiogenic behavior of HUVEC cellcultures. FIG. 13C shows that UNFX-conditioned media attenuates fibrosispathways in epithelial cells. FIG. 13D depicts the positive feedbackloop established by TGFβ1 and Plasminogen Activator Inhibitor-1 (PAI-1).

FIGS. 14A-14B show a Western blot analysis demonstrating the attenuationof fibrosis pathways in mesangial cells.

FIGS. 15A-15C shows that the conditioned media from UNFX containssecreted vesicles. FIG. 15A depicts secreted vesicles, which are bilipidstructures (red) that encompass cytoplasm-derived internal components(green). FIGS. 15B-15C show FACS sorting.

FIG. 16A shows a Western blot in which total protein was prepared andassayed for PAI-1 and bActin. FIG. 16B depicts the microRNA, miR-30b-5p.

FIGS. 17A-17C show representative immunohistochemistry images of PAI-1in Lewis rat kidneys following delivery of bioactive kidney cells afterundergoing a nephrectomy. FIG. 17D shows a comparison of PAI-1expresssion in untreated, nephrectomized rats (red squares), treated,nephrectomized rats (blue diamonds), and control animals (greentriangles). FIG. 17E shows representative Western blot analysis onkidney samples taken at 3 and 6 months post-treatment. FIG. 17F shows a2-hour exposure to NKA conditioned media reduces nuclear localization ofNFκB p65. FIG. 17G depicts the canonical activation of the NFkB pathwayby TNFα.

FIGS. 18A-18B show the nuclear localization of NFkB p65 subunit inanimals with (A) progressive CKD initiated by 5/6 nephrectomy and (B)non-progressive renal insufficiency initiated by unilateral nephrectomy.FIGS. 18C-18D show (C) a Western blot analysis for NFkB p65 in extractsof Lewis rat kidney tissue that have undergone the 5/6 nephrectomy; and(D) electrophoretic mobility shift assay (EMSA) on extracts. FIG. 18Eshows immunohistochemical detection of the NFκB p65 subunit in tissueobtained from Lewis rats with established CKD that received intra-renalinjection of NKA (panel A) or non-bioactive renal cells (panel B).

FIGS. 19A-19C show in vivo evaluation of biomaterials at 1 week and 4weeks post-implantation.

FIGS. 20A-20D show live/dead staining of NKA constructs. FIGS. 20E-20Gshow transcriptomic profiling of NKA constructs.

FIGS. 21A-21B show the secretomic profiling of NKA Constructs.

FIGS. 22A-22B show proteomic profiling of NKA Constructs.

FIGS. 23A-23C show confocal microscopy of NKA Constructs.

FIGS. 24A-24B show in vivo evaluation of NKA Constructs at 1 week and 4weeks post-implantation.

FIGS. 25A-25D show in vivo evaluation of NKA Construct at 8 weekspost-implantation.

FIG. 26 shows conditioned medium from NKA Constructs attenuates TGF-βinduced EMT in HK2 cells in vitro.

FIG. 27 depicts the procedure for exposing cells to low oxygen duringprocessing.

FIG. 28 shows that upon exposure to 2% Oxygen, the following wasobserved: alters distribution of cells across a density gradient,improves overall post-gradient yield

FIG. 29A depicts an assay developed to observe repair of tubularmonolayers in vitro. FIG. 29B shows results of a Quantitative ImageAnalysis (BD Pathway 855 BioImager). FIG. 29C shows cells induced with2% oxygen to be more proficient at repair of tubular epithelialmonolayers.

FIG. 30A depicts an assay developed to observe repair of tubularmonolayers in vitro. FIG. 30B shows that the induction of cells with 2%Oxygen enhanced the migration and wound repair compared to un-induced(21% oxygen). FIG. 30C plots the % of migrated cells against migrationtime.

FIGS. 31A-31B show that osteopontin is secreted by tubular cells and isupregulated in response to injury (Osteopontin Immunocytochemistry:Hoechst nuclear stain (blue), Osteopontin (Red), 10×). Osteopontin isupregulated by injury in established tubular cell monolayers as shown byimmunoflluorescence (FIG. 31A) and ELISA (FIG. 31B).

FIG. 32A shows that the migratory response of cells is mediated in partby osteopontin (Green=migrated cells (5×)). FIG. 32B shows thatneutralizing antibodies (NAb) to osteopontin reduce renal cell migrationresponse by 50%.

FIG. 33 shows that low-oxygen induction of cells modulates expression oftissue remodeling genes.

FIG. 34 depicts a putative mechanism for low oxygen augmentation ofbioactivity of cells leading to renal regeneration.

FIG. 35 shows detection of microvesicles via a Western blot.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to heterogenous mixtures or fractionsof bioactive renal cells (BRCs) and methods of isolating and culturingthe same, as well as methods of treating a subject in need with BRCsand/or BRC-containing constructs formed from a scaffold seeded with BRCsas described herein. The bioactive renal cells may be isolated renalcells including tubular and erythropoietin (EPO)-producing kidney cells.The BRC cell populations may include enriched tubular and EPO-producingcell populations. The BRCs may be derived from or are themselves renalcell fractions from healthy individuals. In addition, the presentinvention provides renal cell fractions obtained from an unhealthyindividual may lack certain cellular components when compared to thecorresponding renal cell fractions of a healthy individual, yet stillretain therapeutic properties. The present invention also providestherapeutically-active cell populations lacking cellular componentscompared to a healthy individual, which cell populations can be, in oneembodiment, isolated and expanded from autologous sources in variousdisease states.

The present invention also relates methods of providing a regenerativeeffect to a native kidney by in vivo contacting the native kidney withproducts secreted by renal cells, as well as methods of preparing thesecreted products. The present invention further relates to the use ofmarkers to determine the presence of renal regeneration followingtreatment with a method described herein.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Principles of TissueEngineering, 3^(rd) Ed. (Edited by R Lanza, R Langer, & J Vacanti), 2007provides one skilled in the art with a general guide to many of theterms used in the present application. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. Indeed, the present invention is in no way limited to themethods and materials described.

The term “cell population” as used herein refers to a number of cellsobtained by isolation directly from a suitable tissue source, usuallyfrom a mammal. The isolated cell population may be subsequently culturedin vitro. Those of ordinary skill in the art will appreciate thatvarious methods for isolating and culturing cell populations for usewith the present invention and various numbers of cells in a cellpopulation that are suitable for use in the present invention. A cellpopulation may be an unfractionated, heterogeneous cell populationderived from the kidney. For example, a heterogeneous cell populationmay be isolated from a kidney biopsy or from whole kidney tissue.Alternatively, the heterogeneous cell population may be derived from invitro cultures of mammalian cells, established from kidney biopsies orwhole kidney tissue. An unfractionated heterogeneous cell population mayalso be referred to as a non-enriched cell population.

The term “native kidney” shall mean the kidney of a living subject. Thesubject may be healthy or un-healthy. An unhealthy subject may have akidney disease.

The term “regenerative effect” shall mean an effect which provides abenefit to a native kidney. The effect may include, without limitation,a reduction in the degree of injury to a native kidney or an improvementin, restoration of, or stabilization of a native kidney function. Renalinjury may be in the form of fibrosis, inflammation, glomerularhypertrophy, etc. and related to kidney disease in the subject.

The term “admixture” as used herein refers to a combination of two ormore isolated, enriched cell populations derived from an unfractionated,heterogeneous cell population. According to certain embodiments, thecell populations of the present invention are renal cell populations.

An “enriched” cell population or preparation refers to a cell populationderived from a starting kidney cell population (e.g., an unfractionated,heterogeneous cell population) that contains a greater percentage of aspecific cell type than the percentage of that cell type in the startingpopulation. For example, a starting kidney cell population can beenriched for a first, a second, a third, a fourth, a fifth, and so on,cell population of interest. As used herein, the terms “cellpopulation”, “cell preparation” and “cell prototype” are usedinterchangeably.

In one aspect, the term “enriched” cell population as used herein refersto a cell population derived from a starting kidney cell population(e.g., a cell suspension from a kidney biopsy or cultured mammaliankidney cells) that contains a percentage of cells capable of producingEPO that is greater than the percentage of cells capable of producingEPO in the starting population. For example, the term “B4” is a cellpopulation derived from a starting kidney cell population that containsa greater percentage of EPO-producing cells, glomerular cells, andvascular cells as compared to the starting population. The cellpopulations of the present invention may be enriched for one or morecell types and depleted of one or more other cell types. For example, anenriched EPO-producing cell population may be enriched for interstitialfibroblasts and depleted of tubular cells and collecting duct epithelialcells relative to the interstitial fibroblasts and tubular cells in anon-enriched cell population, i.e. the starting cell population fromwhich the enriched cell population is derived. In all embodiments citingEPO-enriched or “B4” populations, the enriched cell populations areheterogeneous populations of cells containing cells that can produce EPOin an oxygen-regulated manner, as demonstrated by oxygen-tunable EPOexpression from the endogenous native EPO gene.

In another aspect, an enriched cell population, which contains a greaterpercentage of a specific cell type, e.g., vascular, glomerular, orendocrine cells, than the percentage of that cell type in the startingpopulation, may also lack or be deficient in one or more specific celltypes, e.g., vascular, glomerular, or endocrine cells, as compared to astarting kidney cell population derived from a healthy individual orsubject. For example, the term “B4′,” or B4 prime,” in one aspect, is acell population derived from a starting kidney cell population thatlacks or is deficient in one or more cell types, e.g., vascular,glomerular or endocrine, depending on the disease state of the startingspecimen, as compared to a healthy individual. In one embodiment, theB4′ cell population is derived from a subject having chronic kidneydisease. In one embodiment, the B4′ cell population is derived from asubject having focal segmental glomerulosclerosis (FSGS). In anotherembodiment, the B4′ cell population is derived from a subject havingautoimmune glomerulonephritis. In another aspect, B4′ is a cellpopulation derived from a starting cell population including all celltypes, e.g., vascular, glomerular, or endocrine cells, which is laterdepleted of or made deficient in one or more cell types, e.g., vascular,glomerular, or endocrine cells. In yet another aspect, B4′ is a cellpopulation derived from a starting cell population including all celltypes, e.g., vascular, glomerular, or endocrine cells, in which one ormore specific cell types e.g., vascular, glomerular, or endocrine cells,is later enriched. For example, in one embodiment, a B4′ cell populationmay be enriched for vascular cells but depleted of glomerular and/orendocrine cells. In another embodiment, a B4′ cell population may beenriched for glomerular cells but depleted of vascular and/or endocrinecells. In another embodiment, a B4′ cell population may be enriched forendocrine cells but depleted of vascular and/or glomerular cells. Inanother embodiment, a B4′ cell population may be enriched for vascularand endocrine cells but depleted of glomerular cells. In preferredembodiments, the B4′ cell population, alone or admixed with anotherenriched cell population, e.g., B2 and/or B3, retains therapeuticproperties. A B4′ cell population, for example, is described herein inthe Examples, e.g., Examples 7-9.

In another aspect, an enriched cell population may also refer to a cellpopulation derived from a starting kidney cell population as discussedabove that contains a percentage of cells expressing one or more tubularcell markers that is greater than the percentage of cells expressing oneor more tubular cell markers in the starting population. For example,the term “B2” refers to a cell population derived from a starting kidneycell population that contains a greater percentage of tubular cells ascompared to the starting population. In addition, a cell populationenriched for cells that express one or more tubular cell markers (or“B2”) may contain some epithelial cells from the collecting duct system.Although the cell population enriched for cells that express one or moretubular cell markers (or “B2”) is relatively depleted of EPO-producingcells, glomerular cells, and vascular cells, the enriched population maycontain a smaller percentage of these cells (EPO-producing, glomerular,and vascular) in comparison to the starting population. In general, aheterogeneous cell population is depleted of one or more cell types suchthat the depleted cell population contains a lesser proportion of thecell type(s) relative to the proportion of the cell type(s) contained inthe heterogeneous cell population prior to depletion. The cell typesthat may be depleted are any type of kidney cell. For example, incertain embodiments, the cell types that may be depleted include cellswith large granularity of the collecting duct and tubular system havinga density of < about 1.045 g/ml, referred to as “B1”. In certain otherembodiments, the cell types that may be depleted include debris andsmall cells of low granularity and viabilty having a density of > about1.095 g/ml, referred to as “B5”. In some embodiments, the cellpopulation enriched for tubular cells is relatively depleted of all ofthe following: “B1”, “B5”, oxygen-tunable EPO-expressing cells,glomerular cells, and vascular cells.

The term “hypoxic” culture conditions as used herein refers to cultureconditions in which cells are subjected to a reduction in availableoxygen levels in the culture system relative to standard cultureconditions in which cells are cultured at atmospheric oxygen levels(about 21%). Non-hypoxic conditions are referred to herein as normal ornormoxic culture conditions.

The term “oxygen-tunable” as used herein refers to the ability of cellsto modulate gene expression (up or down) based on the amount of oxygenavailable to the cells. “Hypoxia-inducible” refers to the upregulationof gene expression in response to a reduction in oxygen tension(regardless of the pre-induction or starting oxygen tension).

The term “biomaterial” as used here refers to a natural or syntheticbiocompatible material that is suitable for introduction into livingtissue. A natural biomaterial is a material that is made by a livingsystem. Synthetic biomaterials are materials which are not made by aliving system. The biomaterials disclosed herein may be a combination ofnatural and synthetic biocompatible materials. As used herein,biomaterials include, for example, polymeric matrices and scaffolds.Those of ordinary skill in the art will appreciate that thebiomaterial(s) may be configured in various forms, for example, asliquid hydrogel suspensions, porous foam, and may comprise one or morenatural or synthetic biocompatible materials.

The term “anemia” as used herein refers to a deficit in red blood cellnumber and/or hemoglobin levels due to inadequate production offunctional EPO protein by the EPO-producing cells of a subject, and/orinadequate release of EPO protein into systemic circulation, and/or theinability of erythroblasts in the bone marrow to respond to EPO protein.A subject with anemia is unable to maintain erythroid homeostasis. Ingeneral, anemia can occur with a decline or loss of kidney function(e.g., chronic renal failure), anemia associated with relative EPOdeficiency, anemia associated with congestive heart failure, anemiaassociated with myelo-suppressive therapy such as chemotherapy oranti-viral therapy (e.g., AZT), anemia associated with non-myeloidcancers, anemia associated with viral infections such as HIV, and anemiaof chronic diseases such as autoimmune diseases (e.g., rheumatoidarthritis), liver disease, and multi-organ system failure.

The term “EPO-deficiency” refers to any condition or disorder that istreatable with an erythropoietin receptor agonist (e.g., recombinant EPOor EPO analogs), including anemia.

The term “kidney disease” as used herein refers to disorders associatedwith any stage or degree of acute or chronic renal failure that resultsin a loss of the kidney's ability to perform the function of bloodfiltration and elimination of excess fluid, electrolytes, and wastesfrom the blood. Kidney disease also includes endocrine dysfunctions suchas anemia (erythropoietin-deficiency), and mineral imbalance (Vitamin Ddeficiency). Kidney disease may originate in the kidney or may besecondary to a variety of conditions, including (but not limited to)heart failure, hypertension, diabetes, autoimmune disease, or liverdisease. Kidney disease may be a condition of chronic renal failure thatdevelops after an acute injury to the kidney. For example, injury to thekidney by ischemia and/or exposure to toxicants may cause acute renalfailure; incomplete recovery after acute kidney injury may lead to thedevelopment of chronic renal failure.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures for kidney disease, anemia, EPOdeficiency, tubular transport deficiency, or glomerular filtrationdeficiency wherein the object is to reverse, prevent or slow down(lessen) the targeted disorder. Those in need of treatment include thosealready having a kidney disease, anemia, EPO deficiency, tubulartransport deficiency, or glomerular filtration deficiency as well asthose prone to having a kidney disease, anemia, EPO deficiency, tubulartransport deficiency, or glomerular filtration deficiency or those inwhom the kidney disease, anemia, EPO deficiency, tubular transportdeficiency, or glomerular filtration deficiency is to be prevented. Theterm “treatment” as used herein includes the stabilization and/orimprovement of kidney function.

The term “in vivo contacting” as used herein refers to direct contact invivo between products secreted by an enriched population of renal cells(or an admixture or construct containing renal cells/renal cellfractions) and a native kidney. The direct in vivo contacting may beparacrine, endocrine, or juxtacrine in nature. The products secreted maybe a heterogeneous population of different products described herein.

The term “ribonucleic acid” or “RNA” as used herein refers to a chain ofnucleotide units where each unit is made up of a nitrogenous base, aribose sugar, and a phosphate. The RNA may be in single or doublestanded form. The RNA may be part of, within, or associated with avesicle. The vesicle may be an exosome. RNA includes, withoutlimitation, mRNAs, rRNA, small RNAs, snRNAs, snoRNAs, microRNAs(miRNAs), small interfering RNAs (siRNAs), and noncoding RNAs. The RNAis preferably human RNA.

The term “construct” refers to one or more cell populations deposited onor in a surface of a scaffold or matrix made up of one or more syntheticor naturally-occurring biocompatible materials. The one or more cellpopulations may be coated with, deposited on, embedded in, attached to,seeded, or entrapped in a biomaterial made up of one or more syntheticor naturally-occurring biocompatible polymers, proteins, or peptides.The one or more cell populations may be combined with a biomaterial orscaffold or matrix in vitro or in vivo. In general, the one or morebiocompatible materials used to form the scaffold/biomaterial isselected to direct, facilitate, or permit the formation ofmulticellular, three-dimensional, organization of at least one of thecell populations deposited thereon. The one or more biomaterials used togenerate the construct may also be selected to direct, facilitate, orpermit dispersion and/or integration of the construct or cellularcomponents of the construct with the endogenous host tissue, or todirect, facilitate, or permit the survival, engraftment, tolerance, orfunctional performance of the construct or cellular components of theconstruct.

The term “marker” or “biomarker” refers generally to a DNA, RNA,protein, carbohydrate, or glycolipid-based molecular marker, theexpression or presence of which in a cultured cell population can bedetected by standard methods (or methods disclosed herein) and isconsistent with one or more cells in the cultured cell population beinga particular type of cell. The marker may be a polypeptide expressed bythe cell or an identifiable physical location on a chromosome, such as agene, a restriction endonuclease recognition site or a nucleic acidencoding a polypeptide (e.g., an mRNA) expressed by the native cell. Themarker may be an expressed region of a gene referred to as a “geneexpression marker”, or some segment of DNA with no known codingfunction. The biomarkers may be cell-derived, e.g., secreted, products.

The terms “differentially expressed gene,” “differential geneexpression” and their synonyms, which are used interchangeably, refer toa gene whose expression is activated to a higher or lower level in afirst cell or cell population, relative to its expression in a secondcell or cell population. The terms also include genes whose expressionis activated to a higher or lower level at different stages over timeduring passage of the first or second cell in culture. It is alsounderstood that a differentially expressed gene may be either activatedor inhibited at the nucleic acid level or protein level, or may besubject to alternative splicing to result in a different polypeptideproduct. Such differences may be evidenced by a change in mRNA levels,surface expression, secretion or other partitioning of a polypeptide,for example. Differential gene expression may include a comparison ofexpression between two or more genes or their gene products, or acomparison of the ratios of the expression between two or more genes ortheir gene products, or even a comparison of two differently processedproducts of the same gene, which differ between the first cell and thesecond cell. Differential expression includes both quantitative, as wellas qualitative, differences in the temporal or cellular expressionpattern in a gene or its expression products among, for example, thefirst cell and the second cell. For the purpose of this invention,“differential gene expression” is considered to be present when there isa difference between the expression of a given gene in the first celland the second cell. The differential expression of a marker may be incells from a patient before administration of a cell population,admixture, or construct (the first cell) relative to expression in cellsfrom the patient after administration (the second cell).

The terms “inhibit”, “down-regulate”, “under-express” and “reduce” areused interchangeably and mean that the expression of a gene, or level ofRNA molecules or equivalent RNA molecules encoding one or more proteinsor protein subunits, or activity of one or more proteins or proteinsubunits, is reduced relative to one or more controls, such as, forexample, one or more positive and/or negative controls. Theunder-expression may be in cells from a patient before administration ofa cell population, admixture, or construct relative to cells from thepatient after administration.

The term “up-regulate” or “over-express” is used to mean that theexpression of a gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is elevated relative to oneor more controls, such as, for example, one or more positive and/ornegative controls. The over-expression may be in cells from a patientafter administration of a cell population, admixture, or constructrelative to cells from the patient before administration.

The term “subject” shall mean any single human subject, including apatient, eligible for treatment, who is experiencing or has experiencedone or more signs, symptoms, or other indicators of a kidney disease,anemia, or EPO deficiency. Such subjects include without limitationsubjects who are newly diagnosed or previously diagnosed and are nowexperiencing a recurrence or relapse, or are at risk for a kidneydisease, anemia, or EPO deficiency, no matter the cause. The subject mayhave been previously treated for a kidney disease, anemia, or EPOdeficiency, or not so treated.

The term “patient” refers to any single animal, more preferably a mammal(including such non-human animals as, for example, dogs, cats, horses,rabbits, zoo animals, cows, pigs, sheep, and non-human primates) forwhich treatment is desired. Most preferably, the patient herein is ahuman.

The term “sample” or “patient sample” or “biological sample” shallgenerally mean any biological sample obtained from a subject or patient,body fluid, body tissue, cell line, tissue culture, or other source. Theterm includes tissue biopsies such as, for example, kidney biopsies. Theterm includes cultured cells such as, for example, cultured mammaliankidney cells. Methods for obtaining tissue biopsies and cultured cellsfrom mammals are well known in the art. If the term “sample” is usedalone, it shall still mean that the “sample” is a “biological sample” or“patient sample”, i.e., the terms are used interchangeably.

The term “test sample” refers to a sample from a subject that has beentreated by a method of the present invention. The test sample mayoriginate from various sources in the mammalian subject including,without limitation, blood, semen, serum, urine, bone marrow, mucosa,tissue, etc.

The term “control” or “control sample” refers a negative or positivecontrol in which a negative or positive result is expected to helpcorrelate a result in the test sample. Controls that are suitable forthe present invention include, without limitation, a sample known toexhibit indicators characteristic of normal erythroid homeostasis, asample known to exhibit indicators characteristic of anemia, a sampleobtained from a subject known not to be anemic, and a sample obtainedfrom a subject known to be anemic. Additional controls suitable for usein the methods of the present invention include, without limitation,samples derived from subjects that have been treated withpharmacological agents known to modulate erythropoiesis (e.g.,recombinant EPO or EPO analogs). In addition, the control may be asample obtained from a subject prior to being treated by a method of thepresent invention. An additional suitable control may be a test sampleobtained from a subject known to have any type or stage of kidneydisease, and a sample from a subject known not to have any type or stageof kidney disease. A control may be a normal healthy matched control.Those of skill in the art will appreciate other controls suitable foruse in the present invention.

“Regeneration prognosis”, “regenerative prognosis”, or “prognostic forregeneration” generally refers to a forecast or prediction of theprobable regenerative course or outcome of the administration orimplantation of a cell population, admixture or construct describedherein. For a regeneration prognosis, the forecast or prediction may beinformed by one or more of the following: improvement of a functionalkidney after implantation or administration, development of a functionalkidney after implantation or administration, development of improvedkidney function or capacity after implantation or administration, andexpression of certain markers by the native kidney followingimplantation or administration.

“Regenerated kidney” refers to a native kidney after implantation oradmnistration of a cell population, admixture, or construct as describedherein. The regenerated kidney is characterized by various indicatorsincluding, without limitation, development of function or capacity inthe native kidney, improvement of function or capacity in the nativekidney, and the expression of certain markers in the native kidney.Those of ordinary skill in the art will appreciate that other indicatorsmay be suitable for characterizing a regenerated kidney.

Cell Populations

Isolated, heterogeneous populations of kidney cells, and admixturesthereof, enriched for specific bioactive components or cell types and/ordepleted of specific inactive or undesired components or cell types foruse in the treatment of kidney disease, i.e., providing stabilizationand/or improvement and/or regeneration of kidney function, werepreviously described in U.S. application Ser. No. 12/617,721 filed Nov.12, 2009, the entire contents of which is incorporated herein byreference. The present invention provides isolated renal cell fractionsthat lack cellular components as compared to a healthy individual yetretain therapeutic properties, i.e., provide stabilization and/orimprovement and/or regeneration of kidney function. The cellpopulations, cell fractions, and/or admixtures of cells described hereinmay be derived from healthy individuals, individuals with a kidneydisease, or subjects as described herein.

Bioactive Cell Populations

In one aspect, the present invention is based on the surprising findingthat certain subfractions of a heterogeneous population of renal cells,enriched for bioactive components and depleted of inactive or undesiredcomponents, provide superior therapeutic and regenerative outcomes thanthe starting population. For example, bioactive components of theinvention, e.g., B2, B4, and B3, which are depleted of inactive orundesired components, e.g., B1 and B5, alone or admixed, provideunexpected stabilization and/or improvement and/or regeneration ofkidney function.

In another aspect, the present invention is based on the surprisingfinding that a specific subfraction, B4, depleted of or deficient in oneor more cell types, e.g., vascular, endocrine, or endothelial, i.e.,B4′, retains therapeutic properties, e.g., stabilization and/orimprovement and/or regeneration of kidney function, alone or whenadmixed with other bioactive subfractions, e.g., B2 and/or B3. In apreferred embodiment, the bioactive cell population is B2. In certainembodiments, the B2 cell population is admixed with B4 or B4′. In otherembodiments, the B2 cell population is admixed with B3. In otherembodiments, the B2 cell population is admixed with both B3 and B4, orspecific cellular components of B3 and/or B4.

The B2 cell population is characterized by expression of a tubular cellmarker selected from the group consisting of one or more of thefollowing: megalin, cubilin, hyaluronic acid synthase 2 (HAS2), VitaminD3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogenefamily (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containingion transport regulator 4 (Fxyd4), solute carrier family 9(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3(Aldh1a3), and Calpain-8 (Capn8), and collecting duct marker Aquaporin-4(Aqp4). B2 is larger and more granulated than B3 and/or B4 and thushaving a buoyant density between about 1.045 g/ml and about 1.063 g/ml(rodent), between about 1.045 g/ml and 1.052 g/ml (human), and betweenabout 1.045 g/ml and about 1.058 g/ml (canine).

The B3 cell population is characterized by the expression of vascular,glomerular and proximal tubular markers with some EPO-producing cells,being of an intermediate size and granularity in comparison to B2 andB4, and thus having a buoyant density between about 1.063 g/ml and about1.073 g/ml (rodent), between about 1.052 g/ml and about 1.063 g/ml(human), and between about 1.058 g/ml and about 1.063 g/ml (canine). B3is characterized by expression of markers selected from the groupconsisting of one or more of the following: aquaporin 7 (Aqp7), FXYDdomain-containing ion transport regulator 2 (Fxyd2), solute carrierfamily 17 (sodium phosphate), member 3 (Slc17a3), solute carrier family3, member 1 (Slc3a1), claudin 2 (Cldn2), napsin A aspartic peptidase(Napsa), solute carrier family 2 (facilitated glucose transporter),member 2 (Slc2a2), alanyl (membrane) aminopeptidase (Anpep),transmembrane protein 27 (Tmem27), acyl-CoA synthetase medium-chainfamily member 2 (Acsm2), glutathione peroxidase 3 (Gpx3),fructose-1,6-biphosphatase 1 (Fbp1), and alanine-glyoxylateaminotransferase 2 (Agxt2). B3 is also characterized by the vascularexpression marker Platelet endothelial cell adhesion molecule (Pecam)and the glomerular expression marker podocin (Podn).

The B4 cell population is characterized by the expression of a vascularmarker set containing one or more of the following: PECAM, VEGF, KDR,HIF1a, CD31, CD146; a glomerular marker set containing one or more ofthe following: Podocin (Podn), and Nephrin (Neph); and an oxygen-tunableEPO enriched population compared to unfractionated (UNFX), B2 and B3. B4is also characterized by the expression of one or more of the followingmarkers: chemokine (C-X-C motif) receptor 4 (Cxcr4), endothelin receptortype B (Ednrb), collagen, type V, alpha 2 (Col5a2), Cadherin 5 (Cdh5),plasminogen activator, tissue (Plat), angiopoietin 2 (Angpt2), kinaseinsert domain protein receptor (Kdr), secreted protein, acidic,cysteine-rich (osteonectin) (Sparc), serglycin (Srgn), TIMPmetallopeptidase inhibitor 3 (Timp3), Wilms tumor 1 (Wt1), wingless-typeMMTV integration site family, member 4 (Wnt4), regulator of G-proteinsignaling 4 (Rgs4), Platelet endothelial cell adhesion molecule (Pecam),and Erythropoietin (Epo). B4 is also characterized by smaller, lessgranulated cells compared to either B2 or B3, with a buoyant densitybetween about 1.073 g/ml and about 1.091 g/ml (rodent), between about1.063 g/ml and about 1.091 g/mL (human and canine).

The B4′ cell population is defined as having a buoyant density ofbetween 1.063 g/mL and 1.091 g/mL and expressing one or more of thefollowing markers: PECAM, vEGF, KDR, HIF1a, podocin, nephrin, EPO, CK7,CK8/18/19. In one embodiment, the B4′ cell population is characterizedby the expression of a vascular marker set containing one or more of thefollowing: PECAM, vEGF, KDR, HIF1a, CD31, CD146. In another embodiment,the B4′ cell population is characterized by the expression of anendocrine marker EPO. In one embodiment, the B4′ cell population ischaracterized by the expression of a glomerular marker set containingone or more of the following: Podocin (Podn), and Nephrin (Neph). Incertain embodiments, the B4′ cell population is characterized by theexpression of a vascular marker set containing one or more of thefollowing: PECAM, vEGF, KDR, HIF1a and by the expression of an endocrinemarker EPO. In another embodiment, B4′ is also characterized by smaller,less granulated cells compared to either B2 or B3, with a buoyantdensity between about 1.073 g/ml and about 1.091g/ml (rodent), betweenabout 1.063 g/ml and about 1.091 g/mL (human and canine).

In one aspect, the present invention provides an isolated, enriched B4′population of human renal cells comprising at least one oferythropoietin (EPO)-producing cells, vascular cells, and glomerularcells having a density between 1.063 g/mL and 1.091 g/mL. In oneembodiment, the B4′ cell population is characterized by expression of avascular marker. In certain embodiments, the B4′ cell population is notcharacterized by expression of a glomerular marker. In some embodiments,the B4′ cell population is capable of oxygen-tunable erythropoietin(EPO) expression.

In one embodiment, the B4′ cell population does not include a B2 cellpopulation comprising tubular cells having a density between 1.045 g/mLand 1.052 g/mL. In another embodiment, the B4′ cell population does notinclude a B1 cell population comprising large granular cells of thecollecting duct and tubular system having a density of <1.045 g/ml. Inyet another embodiment, the B4′ cell population does not include a B5cell population comprising debris and small cells of low granularity andviability with a density >1.091 g/ml.

In one embodiment, the B4′ cell population does not include a B2 cellpopulation comprising tubular cells having a density between 1.045 g/mLand 1.052 g/mL; a B1 cell population comprising large granular cells ofthe collecting duct and tubular system having a density of <1.045 g/ml;and a B5 cell population comprising debris and small cells of lowgranularity and viability with a density >1.091 g/ml. In someembodiments, the B4′ cell population may be derived from a subjecthaving kidney disease.

In one aspect, the present invention provides an admixture of humanrenal cells comprising a first cell population, B2, comprising anisolated, enriched population of tubular cells having a density between1.045 g/mL and 1.052 g/mL, and a second cell population, B4′, comprisingerythropoietin (EPO)-producing cells and vascular cells but depleted ofglomerular cells having a density between about 1.063 g/mL and 1.091g/mL, wherein the admixture does not include a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml, or a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In certain embodiment, the B4′ cell population ischaracterized by expression of a vascular marker. In one embodiment, theB4′ cell population is not characterized by expression of a glomerularmarker. In certain embodiments, B2 further comprises collecting ductepithelial cells. In one embodiment, the admixture of cells is capableof receptor-mediated albumin uptake. In another embodiment, theadmixture of cells is capable of oxygen-tunable erythropoietin (EPO)expression. In one embodiment, the admixture contains HAS-2-expressingcells capable of producing and/or stimulating the production ofhigh-molecular weight species of hyaluronic acid (HA) both in vitro andin vivo. In all embodiments, the first and second cell populations maybe derived from kidney tissue or cultured kidney cells.

In one embodiment, the admixture is capable of providing a regenerativestimulus upon in vivo delivery. In other embodiments, the admixture iscapable of reducing the decline of, stabilizing, or improving glomerularfiltration, tubular resorption, urine production, and/or endocrinefunction upon in vivo delivery. In one embodiment, the B4′ cellpopulation is derived from a subject having kidney disease.

In one aspect, the present invention provides an isolated, enriched B4′population of human renal cells comprising at least one oferythropoietin (EPO)-producing cells, vascular cells, and glomerularcells having a density between 1.063 g/mL and 1.091 g/mL. In oneembodiment, the B4′ cell population is characterized by expression of avascular marker. In certain embodiments, the B4′ cell population is notcharacterized by expression of a glomerular marker. The glomerularmarker that is not expressed may be podocin (see Example 7). In someembodiments, the B4′ cell population is capable of oxygen-tunableerythropoietin (EPO) expression.

In one embodiment, the B4′ cell population does not include a B2 cellpopulation comprising tubular cells having a density between 1.045 g/mLand 1.052 g/mL. In another embodiment, the B4′ cell population does notinclude a B1 cell population comprising large granular cells of thecollecting duct and tubular system having a density of <1.045 g/ml. Inyet another embodiment, the B4′ cell population does not include a B5cell population comprising debris and small cells of low granularity andviability with a density >1.091 g/ml.

In one embodiment, the B4′ cell population does not include a B2 cellpopulation comprising tubular cells having a density between 1.045 g/mLand 1.052 g/mL; a B1 cell population comprising large granular cells ofthe collecting duct and tubular system having a density of <1.045 g/ml;and a B5 cell population comprising debris and small cells of lowgranularity and viability with a density >1.091 g/ml.

In some embodiments, the B4′ cell population may be derived from asubject having kidney disease.

In one aspect, the present invention provides an admixture of humanrenal cells comprising a first cell population, B2, comprising anisolated, enriched population of tubular cells having a density between1.045 g/mL and 1.052 g/mL, and a second cell population, B4′, comprisingerythropoietin (EPO)-producing cells and vascular cells but depleted ofglomerular cells having a density between about 1.063 g/mL and 1.091g/mL, wherein the admixture does not include a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml, or a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In certain embodiment, the B4′ cell population ischaracterized by expression of a vascular marker. In one embodiment, theB4′ cell population is not characterized by expression of a glomerularmarker. In certain embodiments, B2 further comprises collecting ductepithelial cells. In one embodiment, the admixture of cells is capableof receptor-mediated albumin uptake. In another embodiment, theadmixture of cells is capable of oxygen-tunable erythropoietin (EPO)expression. In one embodiment, the admixture contains HAS-2-expressingcells capable of producing and/or stimulating the production ofhigh-molecular weight species of hyaluronic acid (HA) both in vitro andin vivo. In all embodiments, the first and second cell populations maybe derived from kidney tissue or cultured kidney cells.

In one embodiment, the admixture is capable of providing a regenerativestimulus upon in vivo delivery. In other embodiments, the admixture iscapable of reducing the decline of, stabilizing, or improving glomerularfiltration, tubular resorption, urine production, and/or endocrinefunction upon in vivo delivery. In one embodiment, the B4′ cellpopulation is derived from a subject having kidney disease.

In a preferred embodiment, the admixture comprises B2 in combinationwith B3 and/or B4. In another preferred embodiment, the admixturecomprises B2 in combination with B3 and/or B4′. In other preferredembodiments, the admixture consists of or consists essentially of (i) B2in combination with B3 and/or B4; or (ii) B2 in combination with B3and/or B4′.

The admixtures that contain a B4′ cell population may contain B2 and/orB3 cell populations that are also obtained from a non-healthy subject.The non-healthy subject may be the same subject from which the B4′fraction was obtained. In contrast to the B4′ cell population, the B2and B3 cell populations obtained from non-healthy subjects are typicallynot deficient in one or more specific cell types as compared to astarting kidney cell population derived from a healthy individual.

Hyaluronic Acid Production by B2 and B4

Hyaluronan (also called hyaluronic acid or hyaluronate) is aglycosaminoglycan (GAG), which consists of a regular repeating sequenceof non-sulfated disaccharide units, specifically N-acetylglucosamine andglucuronic acid. Its molecular weight can range from 400 daltons (thedisaccharide) to over a million daltons. It is found in variable amountsin all tissues, such as the skin, cartilage, and eye, and in most if notall fluids in adult animals. It is especially abundant in early embryos.Space created by hyaluronan, and indeed GAGs in general, permit it toplay a role in cell migration, cell attachment, during wound repair,organogenesis, immune cell adhesion, activation of intracellularsignalling, as well as tumour metastasis. These roles are mediated byspecific protein and proteoglycan binding to Hyaluronan. Cell motilityand immune cell adhesion is mediated by the cell surface receptor RHAMM(Receptor for Hyaluronan-Mediated Motility; Hardwick et al., 1992) andCD44 (Jalkenan et al., 1987; Miyake et al., 1990). Hyaluronan issynthesized directly at the inner membrane of the cell surface with thegrowing polymer extruded through the membrane to the outside of the cellas it is being synthesized. Synthesis is mediated by a single proteinenzyme, hyaluronan synthetase (HAS) whose gene family consists of atleast 3 members.

It has recently been shown that hyaluronic acid interacts with CD44, andsuch interactions may, among other actions, recruit non-resident cells(such as mesenchymal stem cells (MSCs)) to injured renal tissue andenhance renal regeneration (Kidney International (2007) 72, 430-441).

Unexpectedly, it has been found that the B2 and B4 cell preparations arecapable of expressing higher molecular weight species of hyaluronic acid(HA) both in vitro and in vivo, through the actions of hyaluronic acidsynthase-2 (HAS-2)—a marker that is enriched more specifically in the B2cell population. Treatment with B2 in a 5/6 Nx model was shown to reducefibrosis, concomitant with strong expression HAS-2 expression in vivoand the expected production of high-molecular-weight HA within thetreated tissue. Notably, the 5/6 Nx model left untreated resulted infibrosis with limited detection of HAS-2 and little production ofhigh-molecular-weight HA. Without wishing to be bound by theory, it ishypothesized that this anti-inflammatory high-molecular weight speciesof HA produced predominantly by B2 (and to some degree by B4) actssynergystically with the cell preparations in the reduction of renalfibrosis and in the aid of renal regeneration. Accordingly, the instantinvention includes delivery of the cellular prototypes of the inventionin a biomaterial comprising hyaluronic acid. Also comtemplated by theinstant invention is the provision of a biomaterial component of theregenerative stimulus via direct production or stimulation of productionby the implanted cells.

In one aspect, the present invention provides isolated, heterogeneouspopulations of EPO-producing kidney cells for use in the treatment ofkidney disease, anemia and/or EPO deficiency in a subject in need. Inone embodiment, the cell populations are derived from a kidney biopsy.In another embodiment, the cell populations are derived from wholekidney tissue. In one other embodiment, the cell populations are derivedfrom in vitro cultures of mammalian kidney cells, established fromkidney biopsies or whole kidney tissue. In all embodiments, thesepopulations are unfractionated cell populations, also referred to hereinas non-enriched cell populations.

In another aspect, the present invention provides isolated populationsof erythropoietin (EPO)-producing kidney cells that are further enrichedsuch that the proportion of EPO-producing cells in the enrichedsubpopulation is greater relative to the proportion of EPO-producingcells in the starting or initial cell population. In one embodiment, theenriched EPO-producing cell fraction contains a greater proportion ofinterstitial fibroblasts and a lesser proportion of tubular cellsrelative to the interstitial fibroblasts and tubular cells contained inthe unenriched initial population. In certain embodiments, the enrichedEPO-producing cell fraction contains a greater proportion of glomerularcells and vascular cells and a lesser proportion of collecting ductcells relative to the glomerular cells, vascular cells and collectingduct cells contained in the unenriched initial population. In suchembodiments, these populations are referred to herein as the “B4” cellpopulation.

In another aspect, the present invention provides an EPO-producingkidney cell population that is admixed with one or more additionalkidney cell populations. In one embodiment, the EPO-producing cellpopulation is a first cell population enriched for EPO-producing cells,e.g., B4. In another embodiment, the EPO-producing cell population is afirst cell population that is not enriched for EPO-producing cells,e.g., B2. In another embodiment, the first cell population is admixedwith a second kidney cell population. In some embodiments, the secondcell population is enriched for tubular cells, which may be demonstratedby the presence of a tubular cell phenotype. In another embodiment, thetubular cell phenotype may be indicated by the presence of one tubularcell marker. In another embodiment, the tubular cell phenotype may beindicated by the presence of one or more tubular cell markers. Thetubular cell markers include, without limitation, megalin, cubilin,hyaluronic acid synthase 2 (HAS2), Vitamin D3 25-Hydroxylase (CYP2D25),N-cadherin (Ncad), E-cadherin (Ecad), Aquaporin-1 (Aqp1), Aquaporin-2(Aqp2), RAB17, member RAS oncogene family (Rab17), GATA binding protein3 (Gata3), FXYD domain-containing ion transport regulator 4 (Fxyd4),solute carrier family 9 (sodium/hydrogen exchanger), member 4 (Slc9a4),aldehyde dehydrogenase 3 family, member B1 (Aldh3b1), aldehydedehydrogenase 1 family, member A3 (Aldh1a3), and Calpain-8 (Capn8). Inanother embodiment, the first cell population is admixed with at leastone of several types of kidney cells including, without limitation,interstitium-derived cells, tubular cells, collecting duct-derivedcells, glomerulus-derived cells, and/or cells derived from the blood orvasculature. The EPO-producing kidney cell population may contain B4 orB4′ in the form of an admixture with B2 and/or B3, or in the form of anenriched cell population, e.g., B2+B3+B4/B4′.

In one aspect, the EPO-producing kidney cell populations of the presentinvention are characterized by EPO expression and bioresponsiveness tooxygen, such that a reduction in the oxygen tension of the culturesystem results in an induction in the expression of EPO. In oneembodiment, the EPO-producing cell populations are enriched forEPO-producing cells. In one embodiment, the EPO expression is inducedwhen the cell population is cultured under conditions where the cellsare subjected to a reduction in available oxygen levels in the culturesystem as compared to a cell population cultured at normal atmospheric(˜21%) levels of available oxygen. In one embodiment, EPO-producingcells cultured in lower oxygen conditions express greater levels of EPOrelative to EPO-producing cells cultured at normal oxygen conditions. Ingeneral, the culturing of cells at reduced levels of available oxygen(also referred to as hypoxic culture conditions) means that the level ofreduced oxygen is reduced relative to the culturing of cells at normalatmospheric levels of available oxygen (also referred to as normal ornormoxic culture conditions). In one embodiment, hypoxic cell cultureconditions include culturing cells at about less than 1% oxygen, aboutless than 2% oxygen, about less than 3% oxygen, about less than 4%oxygen, or about less than 5% oxygen. In another embodiment, normal ornormoxic culture conditions include culturing cells at about 10% oxygen,about 12% oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen,about 16% oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen,about 20% oxygen, or about 21% oxygen.

In one other embodiment, the induction or increased expression of EPO isobtained and can be observed by culturing cells at about less than 5%available oxygen and comparing EPO expression levels to cells culturedat atmospheric (about 21%) oxygen. In another embodiment, the inductionof EPO is obtained in a culture of cells capable of expressing EPO by amethod that includes a first culture phase in which the culture of cellsis cultivated at atmospheric oxygen (about 21%) for some period of timeand a second culture phase in which the available oxygen levels arereduced and the same cells are cultured at about less than 5% availableoxygen. In another embodiment, the EPO expression that is responsive tohypoxic conditions is regulated by HIF1α. Those of ordinary skill in theart will appreciate that other oxygen manipulation culture conditionsknown in the art may be used for the cells described herein.

In one aspect, the enriched populations of EPO-producing mammalian cellsare characterized by bio-responsiveness (e.g., EPO expression) toperfusion conditions. In one embodiment, the perfusion conditionsinclude transient, intermittent, or continuous fluid flow (perfusion).In one embodiment, the EPO expression is mechanically-induced when themedia in which the cells are cultured is intermittently or continuouslycirculated or agitated in such a manner that dynamic forces aretransferred to the cells via the flow. In one embodiment, the cellssubjected to the transient, intermittent, or continuous fluid flow arecultured in such a manner that they are present as three-dimensionalstructures in or on a material that provides framework and/or space forsuch three-dimensional structures to form. In one embodiment, the cellsare cultured on porous beads and subjected to intermittent or continuousfluid flow by means of a rocking platform, orbiting platform, or spinnerflask. In another embodiment, the cells are cultured onthree-dimensional scaffolding and placed into a device whereby thescaffold is stationary and fluid flows directionally through or acrossthe scaffolding. Those of ordinary skill in the art will appreciate thatother perfusion culture conditions known in the art may be used for thecells described herein.

Inactive Cell Populations

As described herein, the present invention is based, in part, on thesurprising finding that certain subfractions of a heterogeneouspopulation of renal cells, enriched for bioactive components anddepleted of inactive or undesired components, provide superiortherapeutic and regenerative outcomes than the starting population. Inpreferred embodiments, the cellular populations of the instant inventionare depleted of B1 and/or B5 cell populations. For instance, thefollowing may be depleted of B1 and/or B5: admixtures of two or more ofB2, B3, and B4′; an enriched cell population of B2, B3, and B4′.

The B1 cell population comprises large, granular cells of the collectingduct and tubular system, with the cells of the population having abuoyant density less than about 1.045 g/m. The B5 cell population iscomprised of debris and small cells of low granularity and viability andhaving a buoyant density greater than about 1.091 g/ml.

Methods of Isolating and Culturing Cell Populations

The present invention, in one aspect, provides methods for separatingand isolating renal cellular components, e.g., enriched cellpopulations, for therapeutic use, including the treatment of kidneydisease, anemia, EPO deficiency, tubular transport deficiency, andglomerular filtration deficiency. In one embodiment, the cellpopulations are isolated from freshly digested, i.e., mechanically orenzymatically digested, kidney tissue or from heterogeneous in vitrocultures of mammalian kidney cells.

It has unexpectedly been discovered that culturing heterogeneousmixtures of renal cells in hypoxic culture conditions prior toseparation on a density gradient provides for enhanced distribution andcomposition of cells in both B4, including B4′, and B2 and/or B3fractions. The enrichment of oxygen-dependent cells in B4 from B2 wasobserved for renal cells isolated from both diseased and non-diseasedkidneys. Without wishing to be bound by theory, this may be due to oneor more of the following phenomena: 1) selective survival, death, orproliferation of specific cellular components during the hypoxic cultureperiod; 2) alterations in cell granularity and/or size in response tothe hypoxic culture, thereby effecting alterations in buoyant densityand subsequent localization during density gradient separation; and 3)alterations in cell gene / protein expression in response to the hypoxicculture period, thereby resulting in differential characteristics of thecells within any given fraction of the gradient. Thus, in oneembodiment, the cell populations enriched for tubular cells, e.g., B2,are hypoxia-resistant.

Exemplary techniques for separating and isolating the cell populationsof the invention include separation on a density gradient based on thedifferential specific gravity of different cell types contained withinthe population of interest. The specific gravity of any given cell typecan be influenced by the degree of granularity within the cells, theintracellular volume of water, and other factors. In one aspect, thepresent invention provides optimal gradient conditions for isolation ofthe cell preparations of the instant invention, e.g., B2 and B4,including B4′, across multiple species including, but not limited to,human, canine, and rodent. In a preferred embodiment, a density gradientis used to obtain a novel enriched population of tubular cells fraction,i.e., B2 cell population, derived from a heterogeneous population ofrenal cells. In one embodiment, a density gradient is used to obtain anovel enriched population of EPO-producing cells fraction, i.e., B4 cellpopulation, derived from a heterogeneous population of renal cells. Inother embodiments, a density gradient is used to obtain enrichedsubpopulations of tubular cells, glomerular cells, and endothelial cellsof the kidney. In one embodiment, both the EPO-producing and the tubularcells are separated from the red blood cells and cellular debris. In oneembodiment, the EPO-producing, glomerular, and vascular cells areseparated from other cell types and from red blood cells and cellulardebris, while a subpopulation of tubular cells and collecting duct cellsare concomitantly separated from other cell types and from red bloodcells and cellular debris. In one other embodiment, the endocrine,glomerular, and/or vascular cells are separated from other cell typesand from red blood cells and cellular debris, while a subpopulation oftubular cells and collecting duct cells are concomitantly separated fromother cell types and from red blood cells and cellular debris.

The instant invention generated the novel cell populations by using, inpart, the OPTIPREP® (Axis-Shield) density gradient medium, comprising60% nonionic iodinated compound iodixanol in water, based on certain keyfeatures described below. One of skill in the art, however, willrecognize that any density gradient or other means, e.g., immunologicalseparation using cell surface markers known in the art, comprisingnecessary features for isolating the cell populations of the instantinvention may be used in accordance with the invention. It should alsobe recognized by one skilled in the art that the same cellular featuresthat contribute to separation of cellular subpopulations via densitygradients (size and granularity) can be exploited to separate cellularsubpopulations via flow cytometry (forward scatter=a reflection of sizevia flow cytometry, and side scatter=a reflection of granularity).Importantly, the density gradient medium should have low toxicitytowards the specific cells of interest. While the density gradientmedium should have low toxicity toward the specific cells of interest,the instant invention contemplates the use of gradient mediums whichplay a role in the selection process of the cells of interest. Withoutwishing to be bound by theory, it appears that the cell populations ofthe instant invention recovered by the gradient comprising iodixanol areiodixanol-resistant, as there is an appreciable loss of cells betweenthe loading and recovery steps, suggesting that exposure to iodixanolunder the conditions of the gradient leads to elimination of certaincells. The cells appearing in the specific bands after the iodixanolgradient are resistant to any untoward effects of iodixanol and/ordensity gradient exposure. Accordingly, the present invention alsocontemplates the use of additional contrast medias which are mild tomoderate nephrotoxins in the isolation and/or selection of the cellpopulations of the instant invention. In addition, the density gradientmedium should also not bind to proteins in human plasma or adverselyaffect key functions of the cells of interest.

In another aspect, the present invention provides methods of enrichingand/or depleting kidney cell types using fluorescent activated cellsorting (FACS). In one embodiment, kidney cell types may be enrichedand/or depleted using BD FACSAria™ or equivalent.

In another aspect, the present invention provides methods of enrichingand/or depleting kidney cell types using magnetic cell sorting. In oneembodiment, kidney cell types may be enriched and/or depleted using theMiltenyi autoMACS® system or equivalent.

In another aspect, the present invention provides methods ofthree-dimensional culturing of the renal cell populations. In oneaspect, the present invention provides methods of culturing the cellpopulations via continuous perfusion. In one embodiment, the cellpopulations cultured via three-dimensional culturing and continuousperfusion demonstrate greater cellularity and interconnectivity whencompared to cell populations cultured statically. In another embodiment,the cell populations cultured via three dimensional culturing andcontinuous perfusion demonstrate greater expression of EPO, as well asenhanced expression of renal tubule-associate genes such as e-cadherinwhen compared to static cultures of such cell populations.

In yet another embodiment, the cell populations cultured via continuousperfusion demonstrate greater levels of glucose and glutamineconsumption when compared to cell populations cultured statically.

As described herein (including Example 3), low or hypoxic oxygenconditions may be used in the methods to prepare the cell populations ofthe present invention. However, the methods of the present invention maybe used without the step of low oxygen conditioning. In one embodiment,normoxic conditions may be used.

Those of ordinary skill in the art will appreciate that other methods ofisolation and culturing known in the art may be used for the cellsdescribed herein.

Biomaterials (Polymeric Matrices or Scaffolds)

As described in Bertram et al. U.S. Published Application 20070276507(incorporated herein by reference in its entirety), polymeric matricesor scaffolds may be shaped into any number of desirable configurationsto satisfy any number of overall system, geometry or space restrictions.In one embodiment, the matrices or scaffolds of the present inventionmay be three-dimensional and shaped to conform to the dimensions andshapes of an organ or tissue structure. For example, in the use of thepolymeric scaffold for treating kidney disease, anemia, EPO deficiency,tubular transport deficiency, or glomerular filtration deficiency, athree-dimensional (3-D) matrix may be used. A variety of differentlyshaped 3-D scaffolds may be used. Naturally, the polymeric matrix may beshaped in different sizes and shapes to conform to differently sizedpatients. The polymeric matrix may also be shaped in other ways toaccommodate the special needs of the patient. In another embodiment, thepolymeric matrix or scaffold may be a biocompatible, porous polymericscaffold. The scaffolds may be formed from a variety of synthetic ornaturally-occurring materials including, but not limited to, open-cellpolylactic acid (OPLA®), cellulose ether, cellulose, cellulosic ester,fluorinated polyethylene, phenolic, poly-4-methylpentene,polyacrylonitrile, polyamide, polyamideimide, polyacrylate,polybenzoxazole, polycarbonate, polycyanoarylether, polyester,polyestercarbonate, polyether, polyetheretherketone, polyetherimide,polyetherketone, polyethersulfone, polyethylene, polyfluoroolefin,polyimide, polyolefin, polyoxadiazole, polyphenylene oxide,polyphenylene sulfide, polypropylene, polystyrene, polysulfide,polysulfone, polytetrafluoroethylene, polythioether, polytriazole,polyurethane, polyvinyl, polyvinylidene fluoride, regenerated cellulose,silicone, urea-formaldehyde, collagens, laminins, fibronectin, silk,elastin, alginate, hyaluronic acid, agarose, or copolymers or physicalblends thereof. Scaffolding configurations may range from liquidhydrogel suspensions to soft porous scaffolds to rigid, shape-holdingporous scaffolds.

Hydrogels may be formed from a variety of polymeric materials and areuseful in a variety of biomedical applications. Hydrogels can bedescribed physically as three-dimensional networks of hydrophilicpolymers. Depending on the type of hydrogel, they contain varyingpercentages of water, but altogether do not dissolve in water. Despitetheir high water content, hydrogels are capable of additionally bindinggreat volumes of liquid due to the presence of hydrophilic residues.Hydrogels swell extensively without changing their gelatinous structure.The basic physical features of hydrogel can be specifically modified,according to the properties of the polymers used and the additionalspecial equipments of the products.

Preferably, the hydrogel is made of a polymer, a biologically derivedmaterial, a synthetically derived material or combinations thereof, thatis biologically inert and physiologically compatible with mammaliantissues. The hydrogel material preferably does not induce aninflammatory response. Examples of other materials which can be used toform a hydrogel include (a) modified alginates, (b) polysaccharides(e.g. gellan gum and carrageenans) which gel by exposure to monovalentcations, (c) polysaccharides (e.g., hyaluronic acid) that are veryviscous liquids or are thixotropic and form a gel over time by the slowevolution of structure, and (d) polymeric hydrogel precursors (e.g.,polyethylene oxide-polypropylene glycol block copolymers and proteins).U.S. Pat. No. 6,224,893 B1 provides a detailed description of thevarious polymers, and the chemical properties of such polymers, that aresuitable for making hydrogels in accordance with the present invention.

Scaffolding or biomaterial characteristics may enable cells to attachand interact with the scaffolding or biomaterial material, and/or mayprovide porous spaces into which cells can be entrapped. In oneembodiment, the porous scaffolds or biomaterials of the presentinvention allow for the addition or deposition of one or morepopulations or admixtures of cells on a biomaterial configured as aporous scaffold (e.g., by attachment of the cells) and/or within thepores of the scaffold (e.g., by entrapment of the cells). In anotherembodiment, the scaffolds or biomaterials allow or promote for cell:celland/or cell:biomaterial interactions within the scaffold to formconstructs as described herein.

In one embodiment, the biomaterial used in accordance with the presentinvention is comprised of hyaluronic acid (HA) in hydrogel form,containing HA molecules ranging in size from 5.1 kDA to >2×10⁶ kDa. Inanother embodiment, the biomaterial used in accordance with the presentinvention is comprised of hyaluronic acid in porous foam form, alsocontaining HA molecules ranging in size from 5.1 kDA to >2×10⁶ kDa . Inyet another embodiment, the biomaterial used in accordance with thepresent invention is comprised of of a poly-lactic acid (PLA)-basedfoam, having an open-cell structure and pore size of about 50 microns toabout 300 microns. In yet another embodiment, the specific cellpopulations, preferentially B2 but also B4, provide directly and/orstimulate synthesis of high molecular weight Hyaluronic Acid throughHyaluronic Acid Synthase-2 (HAS-2), especially after intra-renalimplantation.

Those of ordinary skill in the art will appreciate that other types ofsynthetic or naturally-occurring materials known in the art may be usedto form scaffolds as described herein.

In one aspect, the present invention provides constructs as describedherein made from the above-referenced scaffolds or biomaterials.

Constructs

In one aspect, the invention provides implantable constructs having oneor more of the cell populations described herein for the treatment ofkidney disease, anemia, or EPO deficiency in a subject in need. In oneembodiment, the construct is made up of a biocompatible material orbiomaterial, scaffold or matrix composed of one or more synthetic ornaturally-occurring biocompatible materials and one or more cellpopulations or admixtures of cells described herein deposited on orembedded in a surface of the scaffold by attachment and/or entrapment.In certain embodiments, the construct is made up of a biomaterial andone or more cell populations or admixtures of cells described hereincoated with, deposited on, deposited in, attached to, entrapped in,embedded in, or combined with the biomaterial component(s). Any of thecell populations described herein, including enriched cell populationsor admixtures thereof, may be used in combination with a matrix to forma construct.

In another embodiment, the deposited cell population or cellularcomponent of the construct is a first kidney cell population enrichedfor oxygen-tunable EPO-producing cells. In another embodiment, the firstkidney cell population contains glomerular and vascular cells inaddition to the oxygen-tunable EPO-producing cells. In one embodiment,the first kidney cell population is a B4′ cell population. In one otherembodiment, the deposited cell population or cellular component(s) ofthe construct includes both the first enriched renal cell population anda second renal cell population. In some embodiments, the second cellpopulation is not enriched for oxygen-tunable EPO producing cells. Inanother embodiment, the second cell population is enriched for renaltubular cells. In another embodiment, the second cell population isenriched for renal tubular cells and contains collecting duct epithelialcells. In other embodiments, the renal tubular cells are characterizedby the expression of one or more tubular cell markers that may include,without limitation, megalin, cubilin, hyaluronic acid synthase 2 (HAS2),Vitamin D3 25-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin(Ecad), Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RASoncogene family (Rab17), GATA binding protein 3 (Gata3), FXYDdomain-containing ion transport regulator 4 (Fxyd4), solute carrierfamily 9 (sodium/hydrogen exchanger), member 4 (Slc9a4), aldehydedehydrogenase 3 family, member B1 (Aldh3b1), aldehyde dehydrogenase 1family, member A3 (Aldh1a3), and Calpain-8 (Capn8).

In one embodiment, the cell populations deposited on or combined withbiomaterials or scaffolds to form constructs of the present inventionare derived from a variety of sources, such as autologous sources.Non-autologous sources are also suitable for use, including withoutlimitation, allogeneic, or syngeneic (autogeneic or isogeneic) sources.

Those of ordinary skill in the art will appreciate there are severalsuitable methods for depositing or otherwise combining cell populationswith biomaterials to form a construct.

In one aspect, the constructs of the present invention are suitable foruse in the methods of use described herein. In one embodiment, theconstructs are suitable for administration to a subject in need oftreatment for a kidney disease of any etiology, anemia, or EPOdeficiency of any etiology. In other embodiments, the constructs aresuitable for administration to a subject in need of an improvement in orrestoration of erythroid homeostasis. In another embodiment, theconstructs are suitable for administration to a subject in need ofimproved kidney function.

In yet another aspect, the present invention provides a construct forimplantation into a subject in need of improved kidney functioncomprising: a) a biomaterial comprising one or more biocompatiblesynthetic polymers or naturally-occurring proteins or peptides; and

b) an admixture of mammalian renal cells derived from a subject havingkidney disease comprising a first cell population, B2, comprising anisolated, enriched population of tubular cells having a density between1.045 g/mL and 1.052 g/mL and a second cell population, B4′, comprisingerythropoietin (EPO)-producing cells and vascular cells but depleted ofglomerular cells having a density between 1.063 g/mL and 1.091 g/mL,coated with, deposited on or in, entrapped in, suspended in, embedded inand/or otherwise combined with the biomaterial. In certain embodiments,the admixture does not include a B1 cell population comprising largegranular cells of the collecting duct and tubular system having adensity of <1.045 g/ml, or a B5 cell population comprising debris andsmall cells of low granularity and viability with a density >1.091 g/ml.

In one embodiment, the construct includes a B4′ cell population which ischaracterized by expression of a vascular marker. In some emodiments,the B4′ cell population is not characterized by expression of aglomerular marker. In certain embodiments, the admixture is capable ofoxygen-tunable erythropoietin (EPO) expression. In all embodiments, theadmixture may be derived from mammalian kidney tissue or cultured kidneycells.

In one embodiment, the construct includes a biomaterial configured as athree-dimensional (3-D) porous biomaterial suitable for entrapmentand/or attachment of the admixture. In another embodiment, the constructincludes a biomaterial configured as a liquid or semi-liquid gelsuitable for embedding, attaching, suspending, or coating mammaliancells. In yet another embodiment, the construct includes a biomaterialconfigured comprised of a predominantly high-molecular weight species ofhyaluronic acid (HA) in hydrogel form. In another embodiment, theconstruct includes a biomaterial comprised of a predominantlyhigh-molecular weight species of hyaluronic acid in porous foam form. Inyet another embodiment, the construct includes a biomaterial comprisedof a poly-lactic acid-based foam having pores of between about 50microns to about 300 microns. In still another embodiment, the constructincludes one or more cell populations that may be derived from a kidneysample that is autologous to the subject in need of improved kidneyfunction. In certain embodiments, the sample is a kidney biopsy. In someemodiments, the subject has a kidney disease. In yet other embodiments,the cell population is derived from a non-autologous kidney sample. Inone embodiment, the construct provides erythroid homeostasis.

Secreted Products

In one other aspect, the present invention concerns products secretedfrom an enriched renal cell population or admixture of enriched renalcell populations, as described herein. In one embodiment, the productsinclude one or more of the following: paracrine factors, endocrinefactors, juxtacrine factors, and vesicles. The vesicles may include oneor more of the following: paracrine factors, endocrine factors,juxtacrine factors, microvesicles, exosomes, and RNA. The secretedproducts may also include products that are not within microvesiclesincluding, without limitation, paracrine factors, endocrine factors,juxtacrine factors, and RNA. For example, extracellular miRNAs have beendetected externally to vesicles (Wang et al., Nuc Acids Res 2010, 1-12doi:10.1093/nar/gkq601, Jul. 7, 2010). The secreted products may also bereferred to as cell-derived products, e.g., cell-derived vesicles.

In one other embodiment, the secreted products may be part of a vesiclederived from renal cells. The vesicles may be capable of delivering thefactors to other destinations. In one embodiment, the vesicles aresecreted vesicles. Several types of secreted vesicles are contemplatedincluding, without limitation, exosomes, microvesicles, ectosomes,membrane particles, exosome-like vesicles, and apoptotic vesicles (Theryet al. 2010. Nat. Rev. Immunol. 9:581-593). In one embodiment, thesecreted vesicles are exosomes. In one other embodiment, the secretedvesicles are microvesicles. In one other embodiment, the secretedvesicles contain or comprise one or more cellular components. Thecomponents may be one or more of the following: membrane lipids, RNA,proteins, metabolities, cytosolic components, and any combinationthereof. In a preferred embodiment, the secreted vesicles comprise,consist of, or consist essentially of microRNAs. Preferably, the miRNAsare human miRNAs. In one embodiment, one or more miRNAs are selectedfrom the group consisting of miR-30b-5p, miR-449a, miR-146a, miR-130a,miR-23b, miR-21, miR-124, and miR-151. In one other embodiment, one ormore miRNAs may be selected from the group consisting of let-7a-1;let-7a-2; let-7a-3; let-7b; let-7c; let-7d; let-7e; let-7f-1; let-7f-2;let-7g; let-7i; mir-1-1; mir-1-2; mir-7-1; mir-7-2; mir-7-3; mir-9-1;mir-9-2; mir-9-3; mir-10a; mir-10b; mir-15a; mir-15b; mir-16-1;mir-16-2; mir-17; mir-18a; mir-18b; mir-19a; mir-19b-1; mir-19b-2;mir-20a; mir-20b; mir-21; mir-22; mir-23a; mir-23b; mir-23c; mir-24-1;mir-24-2; mir-25; mir-26a-1; mir-26a-2; mir-26b; mir-27a; mir-27b;mir-28; mir-29a; mir-29b-1; mir-29b-2; mir-29c; mir-30a; mir-30b;mir-30c-1; mir-30c-2; mir-30d; mir-30e; mir-31; mir-32; mir-33a;mir-33b; mir-34a; mir-34b; mir-34c; mir-92a-1; mir-92a-2; mir-92b;mir-93; mir-95; mir-96; mir-98; mir-99a mir-99b; mir-100; mir-101-1;mir-101-2; mir-103-1; mir-103-1-as; mir-103-2; mir-103-2-as; mir-105-1;mir-105-2; mir-106a; mir-106b; mir-107; mir-122; mir-124-1; mir-124-2;mir-124-3; mir-125a; mir-125b-1; mir-125b-2; mir-126; mir-127;mir-128-1; mir-128-2; mir-129-1; mir-129-2; mir-130a; mir-130b; mir-132;mir-132; mir-133a-1; mir-133a-2; mir-133b; mir-134; mir-135a-1;mir-135a-2; mir-135b; mir-136 MI101351120; mir-137; mir-138-1;mir-138-2; mir-139; mir-140; mir-141; mir-142; mir-143; mir-144;mir-145; mir-146a; mir-146b; mir-147; mir-147b; mir-148a; mir-148b;mir-149; mir-150; mir-151; mir-152; mir-153-1; mir-153-2; mir-154;mir-155; mir-181a-1; mir-181a-2; mir-181b-1; mir-181b-2; mir-181c;mir-181d; mir-182; mir-183; mir-184; mir-185; mir-186; mir-187; mir-188;mir-190; mir-190b; mir-191; mir-192; mir-193a; mir-193b; mir-194-1;mir-194-2; mir-195; mir-196a-1; mir-196a-2; mir-196b; mir-197; mir-198;mir-199a-1; mir-199a-2; mir-199b; mir-200a; mir-200b; mir-200c; mir-202;mir-203; mir-204; mir-205; mir-206; mir-208a; mir-208b; mir-210;mir-211; mir-212; mir-214; mir-215; mir-216a; mir-216b; mir-217;mir-218-1; mir-218-2; mir-219-1; mir-219-2; mir-221; mir-222; mir-223;mir-224; mir-296; mir-297; mir-298; mir-299; mir-300; mir-301a;mir-301b; mir-302a; mir-302b; mir-302c; mir-302d; mir-302e; mir-302f;mir-320a; mir-320b-1; mir-320b-2; mir-320c-1; mir-320c-2; mir-320d-1;mir-320d-2; mir-320e; mir-323; mir-323b; mir-324; mir-325; mir-326;mir-328; mir-329-1; mir-329-2; mir-330; mir-331; mir-335; mir-337;mir-338; mir-339; mir-340; mir-342; mir-345; mir-346; mir-361; mir-362;mir-363; mir-365-1; mir-365-2; mir-367; mir-369; mir-370; mir-37;mir-372; mir-373; mir-374a; mir-374b; mir-374c; mir-375; mir-376a-1;mir-376a-2; mir-376b; mir-376c; mir-377; mir-378; mir-378b; mir-378c;mir-379; mir-380; mir-381; mir-382; mir-383; mir-384; mir-409; mir-410;mir-411; mir-412; mir-421; mir-422a; mir-423; mir-424; mir-425; mir-429;mir-431; mir-432; mir-433; mir-448; mir-449a; mir-449b; mir-449c;mir-450a-1; mir-450a-2; mir-450b; mir-451; mir-452; mir-454; mir-455;mir-466; mir-483; mir-484; mir-485; mir-486; mir-487a; mir-487b;mir-488; mir-489; mir-490; mir-491; mir-492; mir-493; mir-494; mir-495;mir-496; mir-497; mir-498; mir-499; mir-500a; mir-500b; mir-501;mir-502; mir-503; mir-504; mir-505; mir-506; mir-507; mir-508;mir-509-1; mir-509-2; mir-509-3; mir-510; mir-511-1; mir-511-2;mir-512-1; mir-512-2; mir-513a-1; mir-513a-2; mir-513b; mir-513c;mir-514-1; mir-514-2; mir-514-3; mir-514b; mir-515-1; mir-515-2;mir-516a-1; mir-516a-2; mir-516b-1; mir-516b-2; mir-517a; mir-517b;mir-517c; mir-518a-1; mir-518a-2; mir-518b; mir-518c; mir-518d;mir-518e; mir-518f; mir-519a-1; mir-519a-2; mir-519b; mir-519c;mir-519d; mir-519e; mir-520a; mir-520b; mir-520c; mir-520d; mir-520e;mir-520f; mir-520g; mir-520h; mir-521-1; mir-521-2; mir-522; mir-523;mir-524; mir-525; mir-526a-1; mir-526a-2; mir-526b; mir-527; mir-532;mir-539; mir-541; mir-542; mir-543; mir-544; mir-544b; mir-545;mir-548a-1; mir-548a-2; mir-548a-3; mir-548aa-1; mir-548aa-2; mir-548b;mir-548c; mir-548d-1; mir-548d-2; mir-548e; mir-548f-1; mir-548f-2;mir-548f-3; mir-548f-4; mir-548f-5; mir-548g; mir-548h-1; mir-548h-2;mir-548h-3; mir-548h-4; mir-548i-1; mir-548i-2; mir-548i-3; mir-548i-4;mir-548j; mir-548k; mir-5481; mir-548m; mir-548n; mir-548o; mir-548p;mir-548s; mir-548t; mir-548u; mir-548v; mir-548w; mir-548x; mir-548y;mir-548z; mir-549; mir-550a-1; mir-550a-2; mir-550b-1; mir-550b-2;mir-551a; mir-551b; mir-552; mir-553; mir-554; mir-555; mir-556;mir-557; mir-558; mir-559; mir-561; mir-562; mir-563; mir-564; mir-566;mir-567; mir-568; mir-569; mir-570; mir-571; mir-572; mir-573; mir-574;mir-575; mir-576; mir-577; mir-578; mir-579; mir-580; mir-581; mir-582;mir-583; mir-584; mir-585; mir-586; mir-587; mir-588; mir-589; mir-590;mir-591; mir-592; mir-593; mir-595; mir-596; mir-597; mir-598; mir-599;mir-600; mir-601; mir-602; mir-603; mir-604; mir-605; mir-606; mir-607;mir-608; mir-609; mir-610; mir-611; mir-612; mir-613; mir-614; mir-615;mir-616; mir-617; mir-618; mir-619; mir-620; mir-621; mir-622; mir-623;mir-624; mir-625; mir-626; mir-627; mir-628; mir-629; mir-630; mir-631;mir-632; mir-633; mir-634; mir-635; mir-636; mir-637; mir-638; mir-639;mir-640; mir-641; mir-642a; mir-642b; mir-643; mir-644; mir-645;mir-646; mir-647; mir-648; mir-649; mir-650; mir-651; mir-652; mir-653;mir-654; mir-655; mir-656; mir-657; mir-658; mir-659; mir-660; mir-661;mir-662; mir-663; mir-663b; mir-664; mir-665; mir-668; mir-670; mir-671;mir-675; mir-676; mir-708; mir-711; mir-718; mir-720; mir-744; mir-758;mir-759; mir-760; mir-761; mir-762; mir-764; mir-765; mir-766; mir-767;mir-769; mir-770; mir-802; mir-873; mir-874; mir-875; mir-876; mir-877;mir-885; mir-887; mir-888; mir-889; mir-890; mir-891a; mir-891b;mir-892a; mir-892b; mir-920; mir-921; mir-922; mir-924; mir-933;mir-934; mir-935; mir-936; mir-937; mir-938; mir-939; mir-940;mir-941-1; mir-941-2; mir-941-3; mir-941-4; mir-942; mir-942; mir-943;mir-944; mir-1178; mir-1179; mir-1180; mir-1181; mir-1182; mir-1183;mir-1184-1; mir-1184-2; mir-1184-3; mir-1185-1; mir-1185-2; mir-1193;mir-1197; mir-1200; mir-1202; mir-1203; mir-1204; mir-1205; mir-1206;mir-1207; mir-1208; mir-1224; mir-1225; mir-1226; mir-1227; mir-1228;mir-1229; mir-1231; mir-1233-1; mir-1233-2; mir-1234; mir-1236;mir-1237; mir-1238; mir-1243; mir-1244-1; mir-1244-2; mir-1244-3;mir-1245; mir-1246; mir-1247; mir-1248; mir-1249; mir-1250; mir-1251;mir-1252; mir-1253; mir-1254; mir-1255a; mir-1255b-1; mir-1255b-2;mir-1256; mir-1257; mir-1258; mir-1260; mir-1260b; mir-1261; mir-1262;mir-1263; mir-1264; mir-1265; mir-1266; mir-1267; mir-1268; mir-1269;mir-1270-1; mir-1270-2; mir-1271; mir-1272; mir-1273; mir-1273c;mir-1273d; mir-1273e; mir-1274a; mir-1274b; mir-1275; mir-1276;mir-1277; mir-1278; mir-1279; mir-1280; mir-1281; mir-1282; mir-1283-1;mir-1283-2; mir-1284; mir-1285-1; mir-1285-2; mir-1286; mir-1287;mir-1288; mir-1289-1; mir-1289-2; mir-1290; mir-1291; mir-1292;mir-1293; mir-1294; mir-1295; mir-1296; mir-1297; mir-1298; mir-1299;mir-1301; mir-1302-1; mir-1302-10; mir-1302-11; mir-1302-2; mir-1302-3;mir-1302-4; mir-1302-5; mir-1302-6; mir-1302-7; mir-1302-8; mir-1302-9;mir-1303; mir-1304; mir-1305; mir-1306; mir-1307; mir-1321; mir-1322;mir-1323; mir-1324; mir-1468; mir-1469; mir-1470; mir-1471; mir-1537;mir-1538; mir-1539; mir-1825; mir-1827; mir-1908; mir-1909; mir-1910;mir-1911; mir-1912; mir-1913; mir-1914; mir-1915; mir-1972-1;mir-1972-2; mir-1973; mir-1976; mir-2052; mir-2053; mir-2054; mir-2110;mir-2113; mir-2114; mir-2115; mir-2116; mir-2117; mir-2276; mir-2277;mir-2278; mir-2355; mir-2861; mir-2909; mir-3065; mir-3074; mir-3115;mir-3116-1; mir-3116-2; mir-3117; mir-3118-1; mir-3118-2; mir-3118-3;mir-3118-4; mir-3118-5; mir-3118-6; mir-3119-1;mir-3119-2; mir-3120;mir-3121; mir-3122; mir-3123; mir-3124; mir-3125; mir-3126; mir-3127;mir-3128; mir-3129; mir-3130-1; mir-3130-2; mir-3131; mir-3132;mir-3133; mir-3134; mir-3135; mir-3136; mir-3137; mir-3138; mir-3139;mir-3140; mir-3141; mir-3142; mir-3143; mir-3144; mir-3145; mir-3146;mir-3147; mir-3148; mir-3149; mir-3150; mir-3151; mir-3152; mir-3153;mir-3154; mir-3155; mir-3156-1; mir-3156-2; mir-3156-3; mir-3157;mir-3158-1; mir-3158-2; mir-3159; mir-3160-1; mir-3160-2; mir-3161;mir-3162; mir-3163; mir-3164; mir-3165; mir-3166; mir-3167; mir-3168;mir-3169; mir-3170; mir-3171; mir-3173; mir-3174; mir-3175; mir-3176;mir-3177; mir-3178; mir-3179-1; mir-3179-2; mir-3179-3; mir-3180-1;mir-3180-2; mir-3180-3; mir-3180-4; mir-3180-5; mir-3181; mir-3182;mir-3183; mir-3184; mir-3185; mir-3186; mir-3187; mir-3188; mir-3189;mir-3190; mir-3191; mir-3192; mir-3193; mir-3194; mir-3195; mir-3196;mir-3197; mir-3198; mir-3199-1; mir-3199-2; mir-3200; mir-3201;mir-3202-1; mir-3202-2; mir-3605; mir-3606; mir-3607; mir-3609;mir-3610; mir-3611; mir-3612; mir-3613; mir-3614; mir-3615; mir-3616;mir-3617; mir-3618; mir-3619; mir-3620; mir-3621; mir-3622a; mir-3622b;mir-3646; mir-3647; mir-3648; mir-3649; mir-3650; mir-3651; mir-3652;mir-3653; mir-3654; mir-3655; mir-3656mir-3657; mir-3658; mir-3659;mir-3660; mir-3661; mir-3662; mir-3663; mir-3664; mir-3665; mir-3666;mir-3667; mir-3668; mir-3669; mir-3670; mir-3670; mir-3671; mir-3671;mir-3673; mir-3673; mir-3675; mir-3675; mir-3676; mir-3663; mir-3677;mir-3678; mir-3679; mir-3680; mir-3681; mir-3682; mir-3683; mir-3684;mir-3685; mir-3686; mir-3687; mir-3688; mir-3689a; mir-3689b; mir-3690;mir-3691; mir-3692; mir-3713; mir-3714; mir-3907; mir-3908; mir-3909;mir-3910-1; mir-3910-2; mir-3911; mir-3912; mir-3913-1; mir-3913-2;mir-3914-1; mir-3914-2; mir-3915; mir-3916; mir-3917; mir-3918;mir-3919; mir-3920; mir-3921; mir-3922; mir-3923; mir-3924; mir-3925;mir-3926-1; mir-3926-2; mir-3927; mir-3928; mir-3929; mir-3934;mir-3935; mir-3936; mir-3937; mir-3938; mir-3939; mir-3940; mir-3941;mir-3942; mir-3943; mir-3944; mir-3945; mir-4251; mir-4252; mir-4253;mir-4254; mir-4255; mir-4256; mir-4257; mir-4258; mir-4259; mir-4260;mir-4261; mir-4262; mir-4263; mir-4264; mir-4265; mir-4266; mir-4267;mir-4268; mir-4269; mir-4270; mir-4271; mir-4272; mir-4273; mir-4274;mir-4275; mir-4276; mir-4277; mir-4278; mir-4279; mir-4280; mir-4281;mir-4282; mir-4283-1; mir-4283-2; mir-4284; mir-4285; mir-4286;mir-4287; mir-4288; mir-4289; mir-4290; mir-4291; mir-4292; mir-4293;mir-4294; mir-4295; mir-4296; mir-4297; mir-4298; mir-4299; mir-4300;mir-4301; mir-4302; mir-4303; mir-4304; mir-4305; mir-4306; mir-4307;mir-4308; mir-4309; mir-4310; mir-4311; mir-4312; mir-4313; mir-4314;mir-4315-1; mir-4315-2; mir-4316; mir-4317; mir-4318; mir-4319;mir-4320; mir-4321; mir-4322; mir-4323; mir-4324; mir-4325; mir-4326;mir-4327; mir-4328;mir-4329; mir-4329; and mir-4330.

The present invention relates to cell-derived or secreted miRNAsobtainable from the cell populations or constructs described herein. Inone embodiment, one or more of the individual miRNAs may be used toprovide a regenerative effect to a native kidney. Combinations of theindividual miRNAs may be suitable for providing such an effect.Exemplary combinations include two or more of the following: miR-21;miR-23a; miR-30c; miR-1224; miR-23b; miR-92a; miR-100; miR-125b-5p;miR-195; miR-10a-5p; and any combination thereof Another exemplarycombination includes two or more of the following: miR-30b-5p, miR-449a,miR-146a, miR-130a, miR-23b, miR-21, miR-124, miR-151, and anycombination thereof. In one embodiment, the combination of miRNAs mayinclude 2, 3, 4, 5, 6, 7, 8, 9, 10, or more individual miRNAs. Those ofordinary skill in the are will appreciate that other miRNAs andcombinations of mirRNAs may be suitable for use in the presentinvention. Sources of additional miRNAs include miRBase athttp://mirbase.org, which is hosted and maintained in the Faculty ofLife Sciences at the University of Manchester.

In one embodiment, the secreted products comprise paracrine factors. Ingeneral, paracrine factors are molecules synthesized by a cell that candiffuse over small distances to induce or effect changes in aneighboring cell, i.e., a paracrine interaction. The diffusablemolecules are referred to as paracrine factors.

In yet another embodiment, the present invention concerns a compositionof one or more isolated renal-cell derived secreted vesicles, asdescribed herein. Those of ordinary skill in the art will appreciatethat various types of compositions containing the secreted vesicles willbe suitable.

In another aspect, the present invention provides methods of preparingrenal cell secreted products, e.g., vesicles. In one embodiment, themethod includes the steps of providing a renal cell population,including admixtures of one or more enriched renal cell populations. Inanother embodiment, the method further includes the step of culturingthe population under suitable conditions. The conditions may be lowoxygen conditions. In another embodiment, the method further includesthe step of isolating the secreted products from the renal cellpopulation. The secreted vesicles may be obtained from the cell culturemedia of the cell population. In one other embodiment, the renal cellsare characterized by vesicle production and/or secretion that isbioresponsive to oxygen levels, such that a reduction in the oxygentension of the culture system results in an induction of vesicleproduction and/or secretion. In one embodiment, the vesicle productionand/or secretion is induced when the cell population is cultured underconditions where the cells are subjected to a reduction in availableoxygen levels in the culture system as compared to a cell populationcultured at normal atmospheric (˜21%) levels of available oxygen. In oneembodiment, the cell populations cultured in lower oxygen conditionsproduce and/or secrete greater levels of vesicles relative to cellpopulations cultured at normal oxygen conditions. In general, theculturing of cells at reduced levels of available oxygen (also referredto as hypoxic culture conditions) means that the level of reduced oxygenis reduced relative to the culturing of cells at normal atmosphericlevels of available oxygen (also referred to as normal or normoxicculture conditions). In one embodiment, hypoxic cell culture conditionsinclude culturing cells at about less than 1% oxygen, about less than 2%oxygen, about less than 3% oxygen, about less than 4% oxygen, or aboutless than 5% oxygen. In another embodiment, normal or normoxic cultureconditions include culturing cells at about 10% oxygen, about 12%oxygen, about 13% oxygen, about 14% oxygen, about 15% oxygen, about 16%oxygen, about 17% oxygen, about 18% oxygen, about 19% oxygen, about 20%oxygen, or about 21% oxygen. In a preferred embodiment, the methodprovides for the isolation of exosomes and/or microvesicles from renalcells.

In one embodiment, the products are secreted from renal cells. Theproducts may be secreted from renal cells that are not on a scaffold,e.g., the cells are not part of a construct as described herein.

In another embodiment, the products are secreted by renal cells thathave been seeded on a scaffold, e.g., a construct. The constructincludes one or more enriched renal cell populations or an admixturethereof that are directly seeded on or in a scaffold.

In another aspect, the present invention provides in vitro methods forscreening/optimizing/monitoring the biotherapeutic efficacy of one ormore enriched renal cell populations, and admixtures or constructscontaining the same. In one embodiment, the method includes the step ofproviding one or more test populations, test admixture or test construct(the “test article”). In another embodiment, the method includes thestep of culturing the test article under suitable conditions, asdescribed herein. In one other embodiment, the method includes the stepof collecting cell culture media from the cultured test article. Thismedia may be referred to as “conditioned media” and it is expected tocontain products secreted by the renal cells of the test article.

In one other aspect, the conditioned media may be used to conduct one ormore in vitro assays in order to test the biotherapeutic efficacy of thetest article. In one embodiment, the conditioned media is subjected toan epithelial-mesenchymal transition (EMT) assay. The assay may test forEMT induced by TGFβ1. Example 15 provides an exemplary protocol for thisassay.

In another embodiment, the conditioned media is subjected to thedetection of RNAs, e.g., via PCR-based assays, and/or vesicles orexosomes, e.g., via FACS. In one other embodiment, the conditioned mediais subjected to a signaling pathway assay, e.g., immune response (e.g.,NFKB), fibrotic response (PAI-1), and angiogenesis. Examples 12-14provides exemplary protocols for these assays.

Methods of Use

In one aspect, the present invention provides methods for the treatmentof a kidney disease, anemia, or EPO deficiency in a subject in need withthe kidney cell populations and admixtures of kidney cells describedherein. In one embodiment, the method comprises administering to thesubject a composition that includes a first kidney cell populationenriched for EPO-producing cells. In another embodiment, the first cellpopulation is enriched for EPO-producing cells, glomerular cells, andvascular cells. In one embodiment, the first kidney cell population is aB4′ cell population. In another embodiment, the composition may furtherinclude one or more additional kidney cell populations. In oneembodiment, the additional cell population is a second cell populationnot enriched for EPO-producing cells. In another embodiment, theadditional cell population is a second cell population not enriched forEPO-producing cells, glomerular cells, or vascular cells. In anotherembodiment, the composition also includes a kidney cell population oradmixture of kidney cells deposited in, deposited on, embedded in,coated with, or entrapped in a biomaterial to form an implantableconstruct, as described herein, for the treatment of a disease ordisorder described herein. In one embodiment, the cell populations areused alone or in combination with other cells or biomaterials, e.g.,hydrogels, porous scaffolds, or native or synthetic peptides orproteins, to stimulate regeneration in acute or chronic disease states.

In another aspect, the effective treatment of a kidney disease, anemia,or EPO deficiency in a subject by the methods of the present inventioncan be observed through various indicators of erythropoiesis and/orkidney function. In one embodiment, the indicators of erythroidhomeostasis include, without limitation, hematocrit (HCT), hemoglobin(HB), mean corpuscular hemoglobin (MCH), red blood cell count (RBC),reticulocyte number, reticulocyte %, mean corpuscular volume (MCV), andred blood cell distribution width (RDW). In one other embodiment, theindicators of kidney function include, without limitation, serumalbumin, albumin to globulin ratio (A/G ratio), serum phosphorous, serumsodium, kidney size (measurable by ultrasound), serum calcium,phosphorous:calcium ratio, serum potassium, proteinuria, urinecreatinine, serum creatinine, blood nitrogen urea (BUN), cholesterollevels, triglyceride levels and glomerular filtration rate (GFR).Furthermore, several indicators of general health and well-beinginclude, without limitation, weight gain or loss, survival, bloodpressure (mean systemic blood pressure, diastolic blood pressure, orsystolic blood pressure), and physical endurance performance.

In another embodiment, an effective treatment is evidenced bystabilization of one or more indicators of kidney function. Thestabilization of kidney function is demonstrated by the observation of achange in an indicator in a subject treated by a method of the presentinvention as compared to the same indicator in a subject that has notbeen treated by a method of the present invention. Alternatively, thestabilization of kidney function may be demonstrated by the observationof a change in an indicator in a subject treated by a method of thepresent invention as compared to the same indicator in the same subjectprior to treatment. The change in the first indicator may be an increaseor a decrease in value. In one embodiment, the treatment provided by thepresent invention may include stabilization of blood urea nitrogen (BUN)levels in a subject where the BUN levels observed in the subject arelower as compared to a subject with a similar disease state who has notbeen treated by the methods of the present invention. In one otherembodiment, the treatment may include stabilization of serum creatininelevels in a subject where the serum creatinine levels observed in thesubject are lower as compared to a subject with a similar disease statewho has not been treated by the methods of the present invention. Inanother embodiment, the treatment may include stabilization ofhematocrit (HCT) levels in a subject where the HCT levels observed inthe subject are higher as compared to a subject with a similar diseasestate who has not been treated by the methods of the present invention.In another embodiment, the treatment may include stabilization of redblood cell (RBC) levels in a subject where the RBC levels observed inthe subject are higher as compared to a subject with a similar diseasestate who has not been treated by the methods of the present invention.Those of ordinary skill in the art will appreciate that one or moreadditional indicators described herein or known in the art may bemeasured to determine the effective treatment of a kidney disease in thesubject.

In another aspect, the present invention concerns a method of providingerythroid homeostasis in a subject in need. In one embodiment, themethod includes the step of (a) administering to the subject a renalcell population, e.g., B2 or B4′, or admixture of renal cells, e.g.,B2/B4′ and/or B2/B3, as described herein; and (b) determining, in abiological sample from the subject, that the level of an erythropoiesisindicator is different relative to the indicator level in a control,wherein the difference in indicator level (i) indicates the subject isresponsive to the administering step (a), or (ii) is indicative oferythroid homeostasis in the subject. In another embodiment, the methodincludes the step of (a) administering to the subject a compositioncomprising a renal cell population or admixture of renal cells asdescribed herein; and (b) determining, in a biological sample from thesubject, that the level of an erythropoiesis indicator is differentrelative to the indicator level in a control, wherein the difference inindicator level (i) indicates the subject is responsive to theadministering step (s), or (ii) is indicative of erythroid homeostasisin the subject. In another embodiment, the method includes the step of(a) providing a biomaterial or biocompatible polymeric scaffold; (b)depositing a renal cell population or admixture of renal cells of thepresent invention on or within the biomaterial or scaffold in a mannerdescribed herein to form an implantable construct; (c) implanting theconstruct into the subject; and (d) determining, in a biological samplefrom the subject, that the level of an erythropoiesis indicator isdifferent relative to the indicator level in a control, wherein thedifference in indicator level (i) indicates the subject is responsive tothe administering step (a), or (ii) is indicative of erythroidhomeostasis in the subject.

In another aspect, the present invention concerns a method of providingboth stabilization of kidney function and restoration of erythroidhomeostasis to a subject in need, said subject having both a deficit inkidney function and an anemia and/or EPO-deficiency. In one embodiment,the method includes the step of administering a renal cell population oradmixture of renal cells as described herein that contain at least oneof the following cell types: tubular-derived cells, glomerulus-derivedcells, insterstitium-derived cells, collecting duct-derived cells,stromal tissue-derived cells, or cells derived from the vasculature. Inanother embodiment, the population or admixture contains bothEPO-producing cells and tubular epithelial cells, the tubular cellshaving been identified by at least one of the following markers:megalin, cubilin, hyaluronic acid synthase 2 (HAS2), Vitamin D325-Hydroxylase (CYP2D25), N-cadherin (Ncad), E-cadherin (Ecad),Aquaporin-1 (Aqp1), Aquaporin-2 (Aqp2), RAB17, member RAS oncogenefamily (Rab17), GATA binding protein 3 (Gata3), FXYD domain-containingion transport regulator 4 (Fxyd4), solute carrier family 9(sodium/hydrogen exchanger), member 4 (Slc9a4), aldehyde dehydrogenase 3family, member B1 (Aldh3b1), aldehyde dehydrogenase 1 family, member A3(Aldh1a3), and Calpain-8 (Capn8). In this embodiment, treatment of thesubject would be demonstrated by an improvement in at least oneindicator of kidney function concomitant with improvement in at leastone indicator of erythropoiesis, compared to either an untreated subjector to the subject's pre-treatment indicators.

In one aspect, the present invention provides methods of (i) treating akidney disease, anemia, or an EPO-deficiency; (ii) stabilizing kidneyfunction, (iii) restoring erythroid homeostasis, or (iv) any combinationof thereof by administering a renal cell population enriched forEPO-producing cells or admixture of renal cells containing a cellpopulation enriched for EPO-producing cells as described herein, whereinthe beneficial effects of the administration are greater than theeffects of administering a cell population not enriched forEPO-producing cells. In another embodiment, the enriched cell populationprovides an inproved level of serum blood urea nitrogen (BUN). Inanother embodiment, the enriched cell population provides an improvedretention of protein in the serum. In another embodiment, the enrichedcell population provides improved levels of serum cholesterol and/ortriglycerides. In another embodiment, the enriched cell populationprovides an improved level of Vitamin D. In one embodiment, the enrichedcell population provides an improved phosphorus:calcium ratio ascompared to a non-enriched cell population. In another embodiment, theenriched cell population provides an improved level of hemoglobin ascompared to a non-enriched cell population. In a further embodiment, theenriched cell population provides an improved level of serum creatinineas compared to a non-enriched cell population. In yet anotherembodiment, the enriched cell population provides an improved level ofhematocrit as compared to a non-enriched cell population. In a furtherembodiment, the enriched cell population provides an improved level ofred blood cell number (RBC #) as compared to a non-enriched cellpopulation. In one embodiment, the improved level of hematocrit isrestored to 95% normal healthy level. In a further embodiment, theenriched cell population provides an improved reticulocyte number ascompared to a non-enriched cell population. In other embodiments, theenriched cell population provides an improved reticulocyte percentage ascompared to a non-enriched cell population. In yet other embodiments,the enriched cell population provides an improved level of red bloodcell volume distribution width (RDW) as compared to a non-enriched cellpopulation. In yet another embodiment, the enriched cell populationprovides an improved level of hemoglobin as compared to a non-enrichedcell population. In yet another embodiment, the enriched cell populationprovides an erythroietic response in the bone marrow, such that themarrow cellularity is near-normal and the myeloid:erythroid ratio isnear normal.

In another aspect, the present invention provides methods of (i)treating a kidney disease, anemia, or an EPO-deficiency; (ii)stabilizing kidney function, (iii) restoring erythroid homeostasis, or(iv) any combination of thereof by administering an enriched cellpopulation, wherein the beneficial effects of administering a renal cellpopulation or admixture of renal cell populations described herein arecharacterized by improved erythroid homeostasis when compared to thebeneficial effects provided by the administering of recombinant EPO(rEPO). In one embodiment, the population or admixture, whenadministered to a subject in need provides improved erythroidhomeostasis (as determined by hematocrit, hemoglobin, or RBC #) whencompared to the administration of recombinant EPO protein. In oneembodiment, the population or admixture, when administered provides animproved level of hematocrit, RBC, or hemoglobin as compared torecombinant EPO, being no greater than about 10% lower or higher thanhematocrit in a control. In a further embodiment, a single dose ordelivery of the population or admixture, when administered providesimprovement in erythroid homeostasis (as determined by increase inhematocrit, hemoglobin, or RBC #) in the treated subject for a period oftime that significantly exceeds the period of time that a single dose ordelivery of the recombinant EPO protein provides improvement inerythroid homeostasis. In another embodiment, the population oradmixture, when administered at a dose described herein does not resultin hematocrit, hemoglobin, or RBC # greater than about 110% of normallevels in matched healthy controls. In a further embodiment, thepopulation or admixture, when administered at a dose described hereinprovides superior erythroid homeostasis (as determined by hematocrit,hemoglobin, or RBC #) compared to recombinant EPO protein delivered at adose described herein. In another embodiment, the recombinant EPO isdelivered at a dose of about 100 IU/kg, about 200 IU/kg, about 300IU/kg, about 400 IU/kg, or about 500 IU/kg. Those of ordinary skill inthe art will appreciate that other dosages of recombinant EPO known inthe art may be suitable.

Another embodiment of the present invention is directed to the use of atleast one cell population, including enriched cell populations andadmixtures thereof, described herein, or an implantable constructdescribed herein, or secreted products as described herein, for thepreparation of a medicament useful in the treatment of a kidney disease,anemia, or EPO deficiency in a subject in need, the providing oferythroid homeostasis in a subject in need, the improvement of kidneyfunction in a subject in need, or providing a regenerative effect to anative kidney.

Another embodiment of the present invention is directed to the use ofspecific enriched cell population(s) (described herein) for thetreatement of a kidney disease of a specific etiology, based onselection of specific cell subpopulation(s) based on specific verifiedtherapeutic attributes.

In yet another aspect, the present invention provides a method oftreating a kidney disease in a subject in need, comprising:administering to the subject a composition comprising an admixture ofmammalian renal cells comprising a first cell population, B2, comprisingan isolated, enriched population of tubular cells having a densitybetween 1.045 g/mL and 1.052 g/mL, and a second cell population, B4′,comprising erythropoietin (EPO)-producing cells and vascular cells butdepleted of glomerular cells having a density between 1.063 g/mL and1.091 g/mL, wherein the admixture does not include a B1 cell populationcomprising large granular cells of the collecting duct and tubularsystem having a density of <1.045 g/ml, or a B5 cell populationcomprising debris and small cells of low granularity and viability witha density >1.091 g/ml. In certain embodiments, the method includesdetermining in a test sample from the subject that the level of a kidneyfunction indicator is different relative to the indicator level in acontrol, wherein the difference in indicator level is indicative of areduction in decline, a stabilization, or an improvement of one or morekidney functions in the subject. In one embodiment, the B4′ cellpopulation used in the method is characterized by expression of avascular marker. In certain embodiments, the B4′ cell population used inthe method is not characterized by expression of a glomerular marker. Inone embodiment, the admixture of cells used in the method is capable ofoxygen-tunable erythropoietin (EPO) expression. In certain embodiments,the kidney disease to be treated by the methods of the invention isaccompanied by an erythropoietin (EPO) deficiency. In certainembodiments, the EPO deficiency is anemia. In some embodiments, the EPOdeficiency or anemia occurs secondary to renal failure in the subject.In some other embodiments, the EPO deficiency or anemia occurs secondaryto a disorder selected from the group consisting of chronic renalfailure, primary EPO deficiency, chemotherapy or anti-viral therapy,non-myeloid cancer, HIV infection, liver disease, cardiac failure,rheumatoid arthritis, or multi-organ system failure. In certainembodiments, the composition used in the method further comprises abiomaterial comprising one or more biocompatible synthetic polymersand/or naturally-occurring proteins or peptides, wherein the admixtureis coated with, deposited on or in, entrapped in, suspended in, embeddedin and/or otherwise combined with the biomaterial. In certainembodiments, the admixture used in the methods of the invention isderived from mammalian kidney tissue or cultured mammalian kidney cells.In other embodiments, the admixture is derived from a kidney sample thatis autologous to the subject in need. In one embodiment, the sample is akidney biopsy. In other embodiments, the admixture used in the methodsof the invention is derived from a non-autologous kidney sample.

In yet another aspect, the invention provides a use of the cellpreparations and admixtures thereof or an implantable construct of theinstant invention for the preparation of a medicament useful in thetreatment of a kidney disease, anemia or EPO deficiency in a subject inneed thereof.

In another aspect, the present invention provides methods for theregeneration of a native kidney in a subject in need thereof. In oneembodiment, the method includes the step of administering or implantinga cell population, admixture, or construct described herein to thesubject. A regenerated native kidney may be characterized by a number ofindicators including, without limitation, development of function orcapacity in the native kidney, improvement of function or capacity inthe native kidney, and the expression of certain markers in the nativekidney. In one embodiment, the developed or improved function orcapacity may be observed based on the various indicators of erythroidhomeostasis and kidney function described above. In another embodiment,the regenerated kidney is characterized by differential expression ofone or more stem cell markers. The stem cell marker may be one or moreof the following: SRY (sex determining region Y)-box 2 (Sox2);Undifferentiated Embryonic Cell Transcription Factor (UTF1); NodalHomolog from Mouse (NODAL); Prominin 1 (PROM1) or CD133 (CD133); CD24;and any combination thereof. In another embodiment, the expression ofthe stem cell marker(s) is upregulated compared to a control.

The cell populations described herein, including enriched cellpopulations and admixtures thereof as well as constructs containing thesame, may be used to provide a regenerative effect to a native kidney.The effect may be provided by the cells themselves and/or by productssecreted from the cells. The regenerative effect may be characterized byone or more of the following: a reduction in epithelial-mesenchymaltransition (which may be via attenuation of TGF-β signalling); areduction in renal fibrosis; a reduction in renal inflammation;differential expression of a stem cell marker in the native kidney;migration of implanted cells and/or native cells to a site of renalinjury, e.g., tubular injury, engraftment of implanted cells at a siteof renal injury, e.g., tubular injury; stabilization of one or moreindicators of kidney function (as described herein); restoration oferythroid homeostasis (as described herein); and any combination thereof

Methods of Monitoring Regeneration

In another aspect, the present invention provides a prognostic methodfor monitoring regeneration of a native kidney following administrationor implantation of a cell population, admixture, or construct describedherein to the subject. In one embodiment, the method includes the stepof detecting the level of marker expression in a test sample obtainedfrom the subject and in a control sample, wherein a higher level ofexpression of the marker in the test sample, as compared to the controlsample, is prognostic for regeneration of the native kidney in thesubject. In another embodiment, the method includes the detection ofexpression of one or more stem cell markers in the sample. The stem cellmarker may be selected from Sox2; UTF1; NODAL; CD133; CD24; and anycombination thereof. The detecting step may include determining thatexpression of the stem cell marker(s) is upregulated or higher in thetest sample relative to a control sample, wherein the higher level ofexpression is prognostic for regeneration of the subject's nativekidney. In one other embodiment, mRNA expression of the stem cellmarker(s) is detected. In other embodiments, the detection of mRNAexpression may be via a PCR-based method, e.g., qRT-PCR. In situhybridization may also be used for the detection of mRNA expression.

In one other embodiment, polypeptide expression of the stem cellmarker(s) is detected. In another embodiment, polypeptide expression isdetected using an anti-stem cell marker agent. In one other embodiment,the agent is an antibody against the marker. In another embodiment, stemcell marker polypeptide expression is detected usingimmunohistochemistry or a Western Blot.

Those of ordinary skill in the art will appreciate other methods fordetecting mRNA and/or polypeptide expression of markers.

In one embodiment, the detecting step is preceded by the step ofobtaining the test sample from the subject. In another embodiment, thetest sample is kidney tissue.

In one other aspect, the present invention provides the use of markers,such as stem cell markers, as a surrogate marker for regeneration of thenative kidney. Such a marker could be used independent of or inconjunction with an assessment of regeneration based on whether functionor capacity has been developed or improved (e.g., indicators oferythroid homeostasis and kidney function). Monitoring a surrogatemarker over the time course of regeneration may also serve as aprognostic indicator of regeneration.

In another aspect, the invention provides methods for prognosticevaluation of a patient following implantation or administration of acell population, admixture, or construct described herein. In oneembodiment, the method includes the step of detecting the level ofmarker expression in a test sample obtained from said subject; (b)determining the expression level in the test sample relative to thelevel of marker expression relative to a control sample (or a controlreference value); and (c) predicting regenerative prognosis of thepatient based on the determination of marker expression levels, whereina higher level of expression of marker in the test sample, as comparedto the control sample (or a control reference value), is prognostic forregeneration in the subject.

In another aspect, the invention provides methods for prognosticevaluation of a patient following implantation or administration of acell population, admixture, or construct described herein. In oneembodiment, the method includes the steps of (a) obtaining a patientbiological sample; and (b) detecting stem cell marker expression in thebiological sample, wherein stem cell marker expression is prognostic forregeneration of the native kidney in the patient. In some embodiments,increased stem cell marker expression in the patient biological samplerelative to a control sample (or a control reference value) isprognostic for regeneration in the subject. In some embodiments,decreased stem cell marker expression in the patient sample relative tothe control sample (or control reference value) is not prognostic forregeneration in the subject. The patient sample may be a test samplecomprising a biopsy. The patient sample may be a bodily fluid, such asblood or urine.

In one other aspect, the present invention provides prognostic methodsfor monitoring regeneration of a native kidney following administrationor implantation of a cell population, admixture, or construct describedherein to the subject, in which a non-invasive method is used. As analternative to a tissue biopsy, a regenerative outcome in the subjectreceiving treatment can be assessed from examination of a bodily fluid,e.g., urine. It has been discovered that microvesicles obtained fromsubject-derived urine sources contain certain components including,without limitation, specific proteins and miRNAs that are ultimatelyderived from the renal cell populations impacted by treatment with thecell populations of the present invention. These components may includefactors involved in stem cell replication and differentiation,apoptosis, inflammation and immuno-modulation. A temporal analysis ofmicrovesicle-associated miRNA/protein expression patterns allows forcontinuous monitoring of regenerative outcomes within the kidney ofsubjects receiving the cell populations, admixtures, or constructs ofthe present invention. Example 17 describes exemplary protocols foranalysis of the urine of subjects.

These kidney-derived vesicles and/or the luminal contents of kidneyderived vesicles shed into the urine of a subject may be analyzed forbiomarkers indicative of regenerative outcome.

In one embodiment, the present invention provides methods of assessingwhether a kidney disease (KD) patient is responsive to treatment with atherapeutic. The method may include the step of determining or detectingthe amount of vesicles or their luminal contents in a test sampleobtained from a KD patient treated with the therapeutic, as compared toor relative to the amount of vesicles in a control sample, wherein ahigher or lower amount of vesicles or their luminal contents in the testsample as compared to the amount of vesicles or their luminal contentsin the control sample is indicative of the treated patient'sresponsiveness to treatment with the therapeutic.

The present invention also provides a method of monitoring the efficacyof treatment with a therapeutic in a KD patient. In one embodiment, themethod includes the step of determining or detecting the amount ofvesicles in a test sample obtained from a KD patient treated with thetherapeutic, as compared to or relative to the amount of vesicles ortheir luminal contents in a control sample, wherein a higher or loweramount of vesicles or their luminal contents in the test sample ascompared to the amount of vesicles or their luminal contents in thecontrol sample is indicative of the efficacy of treatment with thetherapeutic in the KD patient.

The present invention also provides a method of identifying an agent asa therapeutic effective to treat kidney disease (KD) in a patientsubpopulation. In one embodiment, the method includes the step ofdetermining a correlation between efficacy of the agent and the presenceof an amount of vesicles in samples from the patient subpopulation ascompared to the amount of vesicles or their luminal contents in a sampleobtained from a control sample, wherein a higher or lower amount ofvesicles or their luminal contents in the samples from the patientsubpopulation as compared to the amount of vesicles or their luminalcontents in the control sample is indicative that the agent is effectiveto treat KD in the patient subpopulation.

The present invention provides a method of identifying a patientsubpopulation for which an agent is effective to treat kidney disease(KD). In one embodiment, the method includes the step of determining acorrelation between efficacy of the agent and the presence of an amountof vesicles or their luminal contents in samples from the patientsubpopulation as compared to the amount of vesicles or their luminalcontents in a sample obtained from a control sample, wherein a higher orlower amount of vesicles in the samples from the patient subpopulationas compared to the amount of vesicles or their luminal contents in thecontrol sample is indicative that the agent is effective to treat KD inthe patient subpopulation.

The determining or detecting step may include analyzing the amount ofmiRNA or other secreted products that may exist in the test sample (seeExample 17).

The non-invasive prognostic methods may include the step of obtaining aurine sample from the subject before and/or after administration orimplantation of a cell population, admixture, or construct describedherein. Vesicles and other secreted products may be isolated from theurine samples using standard techniques including without limitation,centriguation to remove unwanted debris (Zhou et al. 2008. Kidney Int.74(5):613-621; Skog et al. U.S. Published Patent Application No.20110053157, each of which is incorporated herein by reference in itsentirety).

The present invention relates to non-invasive methods to detectregenerative outcome in a subject following treatment. The methodsinvolve detection of vesicles or their luminal contents in urine from atreated subject. The luminal contents may be one or more miRNAs. Thedetection of combinations or panels of the individual miRNAs may besuitable for such prognostic methods. Exemplary combinations include twoor more of the following: miR-24; miR-195; miR-871; miR-30b-5p; miR-19b;miR-99a; miR-429; let-7f miR-200a; miR-324-5p; miR-10a-5p; and anycombination thereof. In one embodiment, the combination of miRNAs mayinclude 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more individual miRNAs. Thoseof ordinary skill in the art will appreciate that other miRNAs andcombinations of miRNAs may be suitable for use in such prognosticmethods. Sources of additional miRNAs include miRBase athttp://mirbase.org, which is hosted and maintained in the Faculty ofLife Sciences at the University of Manchester.

Those of skill in the art will appreciate that the prognostic methodsfor detecting regeneration may be suitable for subjects treated withother therapeutics known in the art, apart from the cell populations andconstructs described herein.

In some embodiments, the determining step comprises the use of asoftware program executed by a suitable processor for the purpose of (i)measuring the differential level of marker expression (orvesicles/vesicle contents) in a test sample and a control; and/or (ii)analyzing the data obtained from measuring differential level of markerexpression in a test sample and a control. Suitable software andprocessors are well known in the art and are commercially available. Theprogram may be embodied in software stored on a tangible medium such asCD-ROM, a floppy disk, a hard drive, a DVD, or a memory associated withthe processor, but persons of ordinary skill in the art will readilyappreciate that the entire program or parts thereof could alternativelybe executed by a device other than a processor, and/or embodied infirmware and/or dedicated hardware in a well known manner.

Following the determining step, the measurement results, findings,diagnoses, predictions and/or treatment recommendations are typicallyrecorded and communicated to technicians, physicians and/or patients,for example. In certain embodiments, computers will be used tocommunicate such information to interested parties, such as, patientsand/or the attending physicians. In some embodiments, the assays will beperformed or the assay results analyzed in a country or jurisdictionwhich differs from the country or jurisdiction to which the results ordiagnoses are communicated.

In a preferred embodiment, a prognosis, prediction and/or treatmentrecommendation based on the level of marker expression measured in atest subject having a differential level of marker expression iscommunicated to the subject as soon as possible after the assay iscompleted and the prognosis and/or prediction is generated. The resultsand/or related information may be communicated to the subject by thesubject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, electronic forms of communication, such as email, ortelephone. Communication may be facilitated by use of a computer, suchas in case of email communications. In certain embodiments, thecommunication containing results of a prognostic test and/or conclusionsdrawn from and/or treatment recommendations based on the test, may begenerated and delivered automatically to the subject using a combinationof computer hardware and software which will be familiar to artisansskilled in telecommunications. One example of a healthcare-orientedcommunications system is described in U.S. Pat. No. 6,283,761; however,the present invention is not limited to methods which utilize thisparticular communications system. In certain embodiments of the methodsof the invention, all or some of the method steps, including theassaying of samples, prognosis and/or prediction of regeneration, andcommunicating of assay results or prognoses, may be carried out indiverse (e.g., foreign) jurisdictions.

In another aspect, the prognostic methods described herein provideinformation to an interested party concerning the regenerative successof the implantation or administration.

In all embodiments, the methods of providing a regenerated kidney to asubject in need of such treatment as described herein may include thepost-implantation step of prognostic evaluation of regeneration asdescribed above.

Methods and Routes of Administration

The cell preparations and/or constructs of the instant invention can beadministered alone or in combination with other bioactive components.

The therapeutically effective amount of the renal cell populations oradmixtures of renal cell populations described herein can range from themaximum number of cells that is safely received by the subject to theminimum number of cells necessary for treatment of kidney disease, e.g.,stabilization, reduced rate-of-decline, or improvement of one or morekidney functions. In certain embodiments, the methods of the presentinvention provide the administration of renal cell populations oradmixtures of renal cell populations described herein at a dosage ofabout 10,000 cells/kg, about 20,000 cells/kg, about 30,000 cells/kg,about 40,000 cells/kg, about 50,000 cells/kg, about 100,000 cells/kg,about 200,000 cells/kg, about 300,000 cells/kg, about 400,000 cells/kg,about 500,000 cells/kg, about 600,000 cells/kg, about 700,000 cells/kg,about 800,000 cells/kg, about 900,000 cells/kg, about 1.1×10⁶ cells/kg,about 1.2×10⁶ cells/kg, about 1.3×10⁶ cells/kg, about 1.4×10⁶ cells/kg,about 1.5×10⁶ cells/kg, about 1.6×10⁶ cells/kg, about 1.7×10⁶ cells/kg,about 1.8×10⁶ cells/kg, about 1.9×10⁶ cells/kg, about 2.1×10⁶ cells/kg,about 2.1×10⁶ cells/kg, about 1.2×10⁶ cells/kg, about 2.3×10⁶ cells/kg,about 2.4×10⁶ cells/kg, about 2.5×10⁶ cells/kg, about 2.6×10⁶ cells/kg,about 2.7×10⁶ cells/kg, about 2.8×10⁶ cells/kg, about 2.9×10⁶ cells/kg,about 3×10⁶ cells/kg, about 3.1×10⁶ cells/kg, about 3.2×10⁶ cells/kg,about 3.3×10⁶ cells/kg, about 3.4×10⁶ cells/kg, about 3.5×10⁶ cells/kg,about 3.6×10⁶ cells/kg, about 3.7×10⁶ cells/kg, about 3.8×10⁶ cells/kg,about 3.9×10⁶ cells/kg, about 4×10⁶ cells/kg, about 4.1×10⁶ cells/kg,about 4.2×10⁶ cells/kg, about 4.3×10⁶ cells/kg, about 4.4×10⁶ cells/kg,about 4.5×10⁶ cells/kg, about 4.6×10⁶ cells/kg, about 4.7×10⁶ cells/kg,about 4.8×10⁶ cells/kg, about 4.9×10⁶ cells/kg, or about 5×10⁶ cells/kg.In another embodiment, the dosage of cells to a subject may be a singledosage or a single dosage plus additional dosages. In other embodiments,the dosages may be provided by way of a construct as described herein.In other embodiments, the dosage of cells to a subject may be calculatedbased on the estimated renal mass or functional renal mass.

The therapeutically effective amount of the renal cell populations oradmixtures thereof described herein can be suspended in apharmaceutically acceptable carrier or excipient. Such a carrierincludes, but is not limited to basal culture medium plus 1% serumalbumin, saline, buffered saline, dextrose, water, collagen, alginate,hyaluronic acid, fibrin glue, polyethyleneglycol, polyvinylalcohol,carboxymethylcellulose and combinations thereof. The formulation shouldsuit the mode of administration. Accordingly, the invention provides ause of renal cell populations or admixtures thereof, for example, the B2cell population alone or admixed with the B3 and/or B4 or B4′ cellpopulation, for the manufacture of a medicament to treat kidney diseasein a subject. In some embodiments, the medicament further comprisesrecombinant polypeptides, such as growth factors, chemokines orcytokines. In further embodiments, the medicaments comprise a humankidney-derived cell population. The cells used to manufacture themedicaments can be isolated, derived, or enriched using any of thevariations provided for the methods described herein.

The renal cell preparation(s), or admixtures thereof, or compositionsare formulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for administration to human beings. Typically,compositions for intravenous administration, intra-arterialadministration or administration within the kidney capsule, for example,are solutions in sterile isotonic aqueous buffer. Where necessary, thecomposition can also include a local anesthetic to ameliorate any painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa cryopreserved concentrate in a hermetically sealed container such asan ampoule indicating the quantity of active agent. When the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientscan be mixed prior to administration.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there are a widevariety of suitable formulations of pharmaceutical compositions (see,e.g., Alfonso R Gennaro (ed), Remington: The Science and Practice ofPharmacy, formerly Remington's Pharmaceutical Sciences 20th ed.,Lippincott, Williams & Wilkins, 2003, incorporated herein by referencein its entirety). The pharmaceutical compositions are generallyformulated as sterile, substantially isotonic and in full compliancewith all Good Manufacturing Practice (GMP) regulations of the U.S. Foodand Drug Administration.

One aspect of the invention further provides a pharmaceuticalformulation, comprising a renal cell preparation of the invention, forexample, the B2 cell preparation alone or incombination with the B3and/or B4 or B4′ cell preparation, and a pharmaceutically acceptablecarrier. In some embodiments, the formulation comprises from 10⁴ to 10⁹mammalian kidney-derived cells.

In one aspect, the present invention provides methods of providing oneor more of the cell populations described herein, including admixtures,to a subject in need. In one embodiment, the source of the cellpopulation(s) may be autologous or allogeneic, syngeneic (autogeneic orisogeneic), and any combination thereof. In instances where the sourceis not autologous, the methods may include the administration of animmunosuppressant agent. Suitable immunosuppressant drugs include,without limitation, azathioprine, cyclophosphamide, mizoribine,ciclosporin, tacrolimus hydrate, chlorambucil, lobenzarit disodium,auranofin, alprostadil, gusperimus hydrochloride, biosynsorb, muromonab,alefacept, pentostatin, daclizumab, sirolimus, mycophenolate mofetil,leflonomide, basiliximab, dornase a, bindarid, cladribine, pimecrolimus,ilodecakin, cedelizumab, efalizumab, everolimus, anisperimus,gavilimomab, faralimomab, clofarabine, rapamycin, siplizumab, saireito,LDP-03, CD4, SR-43551, SK&F-106615, IDEC-114, IDEC-131, FTY-720,TSK-204, LF-080299, A-86281, A-802715, GVH-313, HMR-1279, ZD-7349,IPL-423323, CBP-1011, MT-1345, CNI-1493, CBP-2011, J-695, LJP-920,L-732531, ABX-RB2, AP-1903, IDPS, BMS-205820, BMS-224818, CTLA4-1g,ER-49890, ER-38925, ISAtx-247, RDP-58, PNU-156804, LJP-1082, TMC-95A,TV-4710, PTR-262-MG, and AGI-1096 (see U.S. Pat. No. 7,563,822). Thoseof ordinary skill in the art will appreciate other suitableimmunosuppressant drugs.

The treatment methods of the subject invention involve the delivery ofan isolated renal cell population, or admixture thereof, intoindividuals. In one embodiment, direct administration of cells to thesite of intended benefit is preferred. In one embodiment, the cellpreparations, or admixtures thereof, of the instant invention aredelivered to an individual in a delivery vehicle.

A subject in need may also be treated by in vivo contacting of a nativekidney with products secreted from one or more enriched renal cellpopulations, and/or an admixture or construct containing the same. Thestep of contacting a native kidney in vivo with secreted products may beaccomplished through the use/administration of a population of secretedproducts from cell culture media, e.g., conditioned media, or byimplantation of an enriched cell population, and admixture, or aconstruct capable of secreting the products in vivo. The step of in vivocontacting provides a regenerative effect to the native kidney.

A variety of means for administering cells and/or secreted products tosubjects will, in view of this specification, be apparent to those ofskill in the art. Such methods include injection of the cells into atarget site in a subject. Cells and/or secreted products can be insertedinto a delivery device or vehicle, which facilitates introduction byinjection or implantation into the subjects. In certain embodiments, thedelivery vehicle can include natural materials. In certain otherembodiments, the delivery vehicle can include synthetic materials. Inone embodiment, the delivery vehicle provides a structure to mimic orappropriately fit into the organ's architecture. In other embodiments,the delivery vehicle is fluid-like in nature. Such delivery devices caninclude tubes, e.g., catheters, for injecting cells and fluids into thebody of a recipient subject. In a preferred embodiment, the tubesadditionally have a needle, e.g., a syringe, through which the cells ofthe invention can be introduced into the subject at a desired location.In some embodiments, mammalian kidney-derived cell populations areformulated for administration into a blood vessel via a catheter (wherethe term “catheter” is intended to include any of the various tube-likesystems for delivery of substances to a blood vessel). Alternatively,the cells can be inserted into or onto a biomaterial or scaffold,including but not limited to textiles, such as weaves, knits, braids,meshes, and non-wovens, perforated films, sponges and foams, and beads,such as solid or porous beads, microparticles, nanoparticles, and thelike (e.g., Cultispher-S gelatin beads -Sigma). The cells can beprepared for delivery in a variety of different forms. For example, thecells can be suspended in a solution or gel. Cells can be mixed with apharmaceutically acceptable carrier or diluent in which the cells of theinvention remain viable. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid, and will often beisotonic. Preferably, the solution is stable under the conditions ofmanufacture and storage and preserved against the contaminating actionof microorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. One of skill in the art will appreciate that the deliveryvehicle used in the delivery of the cell populations and admixturesthereof of the instant invention can include combinations of theabove-mentioned characteristics.

Modes of administration of the isolated renal cell population(s), forexample, the B2 cell population alone or admixed with B4′ and/or B3,include, but are not limited to, systemic, intra-renal (e.g.,parenchymal), intravenous or intra-arterial injection and injectiondirectly into the tissue at the intended site of activity. Additionalmodes of administration to be used in accordance with the presentinvention include single or multiple injection(s) via direct laparotomy,via direct laparoscopy, transabdominal, or percutaneous. Still yetadditional modes of administration to be used in accordance with thepresent invention include, for example, retrograde and ureteropelvicinfusion. Surgical means of administration include one-step proceduressuch as, but not limited to, partial nephrectomy and constructimplantation, partial nephrectomy, partial pyelectomy, vascularizationwith omentum peritoneum, multifocal biopsy needle tracks, cone orpyramidal, to cylinder, and renal pole-like replacement, as well astwo-step procedures including, for example, organoid-internal bioreactorfor replanting. In one embodiment, the admixtures of cells are deliveredvia the same route at the same time. In another embodiment, each of thecell compositions comprising the controlled admixture are deliveredseparately to specific locations or via specific methodologies, eithersimultaneously or in a temporally-controlled manner, by one or more ofthe methods described herein.

The appropriate cell implantation dosage in humans can be determinedfrom existing information relating to either the activity of the cells,for example EPO production, or extrapolated from dosing studiesconducted in preclinical studies. From in vitro culture and in vivoanimal experiments, the amount of cells can be quantified and used incalculating an appropriate dosage of implanted material. Additionally,the patient can be monitored to determine if additional implantation canbe made or implanted material reduced accordingly.

One or more other components can be added to the cell populations andadmixtures thereof of the instant invention, including selectedextracellular matrix components, such as one or more types of collagenor hyaluronic acid known in the art, and/or growth factors,platelet-rich plasma and drugs.

Those of ordinary skill in the art will appreciate the variousformulations and methods of adminstration suitable for the secretedproducts described herein.

Kits

The instant invention further includes kits comprising the polymericmatrices and scaffolds of the invention and related materials, and/orcell culture media and instructions for use. The instructions for usemay contain, for example, instructions for culture of the cells oradministration of the cells and/or cell products. In one embodiment, thepresent invention provides a kit comprising a scaffold as describedherein and instructions. In yet another embodiment, the kit includes anagent for detection of marker expression, reagents for use of the agent,and instructions for use. This kit may be used for the purpose ofdeterming the regenerative prognosis of a native kidney in a subjectfollowing the implantation or administration of a cell population, anadmixture, or a construct described herein. The kit may also be used todetermine the biotherapeutic efficacy of a cell population, admixture,or construct described herein.

Reports

The methods of this invention, when practiced for commercial purposesgenerally produce a report or summary of the regenerative prognosis. Themethods of this invention will produce a report comprising a predictionof the probable course or outcome of regeneration before and after anyadministration or implantation of a cell population, an admixture, or aconstruct described herein. The report may include information on anyindicator pertinent to the prognosis. The methods and reports of thisinvention can further include storing the report in a database.Alternatively, the method can further create a record in a database forthe subject and populate the record with data. In one embodiment thereport is a paper report, in another embodiment the report is anauditory report, in another embodiment the report is an electronicrecord. It is contemplated that the report is provided to a physicianand/or the patient. The receiving of the report can further includeestablishing a network connection to a server computer that includes thedata and report and requesting the data and report from the servercomputer. The methods provided by the present invention may also beautomated in whole or in part.

All patents, patent applications, and literature references cited in thepresent specification are hereby incorporated by reference in theirentirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

EXAMPLES Example 1—Isolation & Characterization of Bioresponsive RenalCells

A case of idiopathic progressive chronic kidney disease (CKD) withanemia in an adult male swine (Sus scrofa) provided fresh diseasedkidney tissue for the assessment of cellular composition andcharacterization with direct comparison to age-matched normal swinekidney tissue. Histological examination of the kidney tissue at the timeof harvest confirmed renal disease characterized by severe diffusechronic interstitial fibrosis and crescentic glomerulonephritis withmultifocal fibrosis. Clinical chemistry confirmed azotemia (elevation ofblood urea nitrogen and serum creatinine), and mild anemia (mildreduction in hematocrit and depressed hemoglobin levels). Cells wereisolated, expanded, and characterized from both diseased and normalkidney tissue. As shown in FIG. 1 of Presnell et al. WO/2010/056328(incorporated herein by reference in its entirety), a Gomori's Trichromestain highlighs the fibrosis (blue staining indicated by arrows) in thediseased kidney tissue compared to the normal kidney tissue. Functionaltubular cells, expressing cubulin:megalin and capable ofreceptor-mediated albumin transport, were propagated from both normaland diseased kidney tissue. Erythropoietin (EPO)-expressing cells werealso present in the cultures and were retained through multiple passagesand freeze/thaw cycles. Furthermore, molecular analyses confirmed thatthe EPO-expressing cells from both normal and diseased tissue respondedto hypoxic conditions in vitro with HIF1α-driven induction of EPO andother hypoxia-regulated gene targets, including vEGF. Cells wereisolated from the porcine kidney tissue via enzymatic digestion withcollagenase+dispase, and were also isolated in separate experiments byperforming simple mechanical digestion and explant culture. At passagetwo, explant-derived cell cultures containing epo-expressing cells weresubjected to both atmospheric (21%) and varying hypoxic (<5%) cultureconditions to determine whether exposure to hypoxia culminated inupregulation of EPO gene expression. As noted with rodent cultures (seeExample 3), the normal pig displayed oxygen-dependent expression andregulation of the EPO gene. Surprisingly, despite the uremic / anemicstate of the CKD pig (Hematocrit <34, Creatinine >9.0) EPO expressingcells were easily isolated and propagated from the tissue and expressionof the EPO gene remained hypoxia regulated, as shown in FIG. 2 of ofPresnell et al. WO/2010/056328 (incorporated herein by reference in itsentirety). As shown in FIG. 3 of Presnell et al. WO/2010/056328(incorporated herein by reference in its entirety), cells in thepropagated cultures demonstrated the ability to self-organize intotubule-like structures. As shown in FIG. 4 of Presnell et al.WO/2010/056328 (incorporated herein by reference in its entirety), thepresence of functional tubular cells in the culture (at passage 3) wasconfirmed by observing receptor-mediated uptake of FITC-conjugatedAlbumin by the cultured cells. The green dots (indicated by thin whitearrows) represent endocytosed fluorescein-conjugated albumin which ismediated by tubular cell-specific receptors, Megalin and Cubilin,indicating protein reabosroption by functional tubular cells. The bluestaining (indicated by thick white arrows) is Hoescht-stained nuclei.Taken together, these data suggest that functional tubular and endocrinecells can be isolated and propagated from porcine renal tissues, even inrenal tissues that have been severely compromised with CKD. Furthermore,these findings support the advancement of autologous cell-basedtherapeutic products for the treatment of CKD.

In addition, EPO-producing cells were isolated enzymatically from normaladult human kidney (as described above in Example 1). As shown in FIG. 5of Presnell et al. WO/2010/056328 (incorporated herein by reference inits entirety), the isolation procedure resulted in more relative EPOexpression after isolation than in the initial tissue. As shown in FIG.6 of Presnell et al. WO/2010/056328 (incorporated herein by reference inits entirety), it is possible to maintain the human EPO producing cellsin culture with retention of EPO gene expression. Human cells werecultured/propagated on plain tissue-culture treated plastic or plasticthat had been coated with some extracellular matrix, such as, forinstance, fibronectin or collagen, and all were found to support EPOexpression over time.

Example 2—Isolation & Enrichment of Specific Bioreactive Renal Cells

Kidney cell isolation: Briefly, batches of 10, 2-week-old male Lewis ratkidneys were obtained from a commercial supplier (Hilltop Lab AnimalsInc.) and shipped overnight in Viaspan preservation medium at atemperature around 4° C. All steps described herein were carried out ina biological safety cabinet (BSC) to preserve sterility. The kidneyswere washed in Hank's balanced salt solution (HBSS) 3 times to rinse outthe Viaspan preservation medium. After the third wash the remainingkidney capsules were removed as well as any remaining stromaltissue. Themajor calyx was also removed using micro dissection techniques. Thekidneys were then finely minced into a slurry using a sterile scalpel.The slurry was then transferred into a 50 ml conical centrifuge tube andweighed. A small sample was collected for RNA and placed into anRNAse-free sterile 1.5 ml micro-centrifuge tube and snap frozen inliquid nitrogen. Once frozen, it was then transferred to the −80 degreefreezer until analysis. The tissue weight of 10 juvenile kidneys equaledapproximately 1 gram. Based on the weight of the batch, the digestionmedium was adjusted to deliver 20 mls of digestion medium per 1 gram oftissue. Digestion buffer for this procedure contained 4 Units of Dispase1(Stem Cell Tech) in HBSS, 300 Units/ml of Collagenase type IV(Worthington) with 5 mM CaCl₂ (Sigma).

The appropriate volume of pre-warmed digestion buffer was added to thetube, which was then sealed and placed on a rocker in a 37° C. incubatorfor 20 minutes. This first digestion step removes many red blood cellsand enhances the digestion of the remaining tissue. After 20 minutes,the tube was removed and placed in the BSC. The tissue was allowed tosettle at the bottom of the tube and then the supernatant was removed.The remaining tissue was then supplemented with fresh digestion bufferequaling the starting volume. The tube was again placed on a rocker in a37° C. incubator for an additional 30 minutes.

After 30 minutes the digestion mixture was pipetted through a 70 μm cellstrainer (BD Falcon) into an equal volume of neutralization buffer (DMEMw/10% FBS) to stop the digestion reaction. The cell suspension was thenwashed by centrifugation at 300×g for 5 min. After centrifugation, thepellet was then re-suspended in 20 mls KSFM medium and a sample acquiredfor cell counting and viability assessment using trypan blue exclusion.Once the cell count was calculated, 1 million cells were collected forRNA, washed in PBS, and snap frozen in liquid nitrogen. The remainingcell suspension was brought up to 50 mls with KSFM medium and washedagain by centrifugation at 300×g for 5 minutes. After washing, the cellpellet was re-suspended in a concentration of 15 million cells per ml ofKSFM.

Five milliliters of kidney cell suspension were then added to 5 mls of30% (w/v) Optiprep® in 15 ml conical centrifuge tubes (BD Falcon) andmixed by inversion 6 times. This formed a final mixture of 15% (w/v) ofOptiprep®. Post inversion, tubes were carefully layered with 1 mL PBS.The tubes were centrifuged at 800×g for 15 minutes without brake. Aftercentrifugation, the tubes were removed and a cell band was formed at thetop of the mixing gradient. There was also a pellet containing red bloodcells, dead cells, and a small population of live cells that includedsome small less granular cells, some epo-producing cells, some tubularcells, and some endothelial cells. The band was carefully removed usinga pipette and transferred to another 15 ml conical tube. The gradientmedium was removed by aspiration and the pellet was collected byre-suspension in 1 ml KSFM. The band cells and pellet cells were thenrecombined and re-suspended in at least 3 dilutions of the collectedband volume using KSFM and washed by centrifugation at 300×g for 5minutes. Post washing, the cells were re-suspended in 20 mls of KSFM anda sample for cell counting was collected. Once the cell count wascalculated using trypan blue exclusion, 1 million cells were collectedfor an RNA sample, washed in PBS, and snap frozen in liquid nitrogen.

Pre-Culture ‘Clean-up’ to enhance viability and culture performance ofSpecific Bioactive Renal Cells Using Density Gradient Separation: Toyield a clean, viable population of cells for culture, a cell suspensionwas first generated as described above in “Kidney Cell Isolation”. As anoptional step and as a means of cleaning up the initial preparation, upto 100 million total cells, suspended in sterile isotonic buffer weremixed thoroughly 1:1 with an equal volume of 30% Optiprep® prepared atroom temperature from stock 60% (w/v) iodixanol (thus yielding a final15% w/v Optiprep solution) and mixed thoroughly by inversion six times.After mixing, 1 ml PBS buffer was carefully layered on top of the mixedcell suspension. The gradient tubes were then carefully loaded into thecentrifuge, ensuring appropriate balance. The gradient tubes werecentrifuged at 800×g for 15 minutes at 25° C. without brake. Thecleaned-up cell population (containing viable and functional collectingduct, tubular, endocrine, glomerular, and vascular cells) segmentedbetween 6% and 8% (w/v) Optiprep®, corresponding to a density between1.025-1.045 g/mL. Other cells and debris pelleted to the bottom of thetube.

Kidney Cell Culture: The combined cell band and pellet were then platedin tissue culture treated triple flasks (Nunc T500) or equivalent at acell concentration of 30,000 cells per cm2 in 150 mls of a 50:50 mixtureof DMEM(high glucose)/KSFM containing 5% (v/v)FBS, 2.5 μg EGF, 25 mgBPE, 1X ITS (insulin/transferrin/sodium selenite medium supplement) withantibiotic/antimycotic. The cells were cultured in a humidified 5% CO2incubator for 2-3 days, providing a 21% atmospheric oxygen level for thecells. After two days, the medium was changed and the cultures wereplaced in 2% oxygen-level environment provided by a CO2/Nitrogen gasmultigas humidified incubator (Sanyo) for 24 hrs. Following the 24 hrincubation, the cells were washed with 60 mls of 1XPBS and then removedusing 40 mls 0.25% (w/v) trypsin/EDTA (Gibco). Upon removal, the cellsuspension was neutralized with an equal volume of KSFM containing 10%FBS. The cells were then washed by centrifugation 300×g for 10 minutes.After washing, the cells were re-suspended in 20 mls of KSFM andtransferred to a 50 ml conical tube and a sample was collected for cellcounting. Once the viable cell count was determined using trypan blueexclusion, 1 million cells were collected for an RNA sample, washed inPBS, and snap frozen in liquid nitrogen. The cells were washed again inPBS and collected by centrifugation at 300×g for 5 minutes. The washedcell pellet was re-suspended in KSFM at a concentration of 37.5 millioncells/ml.

Enriching for Specific Bioactive Renal Cells Using Density Step GradientSeparation: Cultured kidney cells, predominantly composed of renaltubular cells but containing small subpopulations of other cell types(collecting duct, glomerular, vascular, and endocrine) were separatedinto their component subpopulations using a density step gradient madefrom multiple concentrations w/v of iodixanol (Optiprep). The cultureswere placed into a hypoxic environment for up to 24 hours prior toharvest and application to the gradient. A stepped gradient was createdby layering four different density mediums on top of each other in asterile 15 mL conical tube, placing the solution with the highestdensity on the bottom and layering to the least dense solution on thetop. Cells were applied to the top of the step gradient and centrifuged,which resulted in segregation of the population into multiple bandsbased on size and granularity.

Briefly, densities of 7, 11, 13, and 16% Optiprep® (60% w/v Iodixanol)were made using KFSM medium as diluents. For example: for 50 mls of7%(w/v) Optiprep®, 5.83 mls of stock 60% (w/v) Iodixanol was added to44.17 mls of KSFM medium and mixed well by inversion. A peristaltic pump(Master Flex L/S) loaded with sterile L/S 16 Tygon tubing connected tosterile capillary tubes was set to a flow rate of 2 ml per minute, and 2mL of each of the four solutions was loaded into a sterile conical 15 mLtube, beginning with the 16% solution, followed by the 13% solution, the11% solution, and the 7% solution. Finally, 2 mL of cell suspensioncontaining 75 million cultured rodent kidney cells was loaded atop thestep gradient (suspensions having been generated as described above in‘Kidney cell Culture’). Importantly, as the pump was started to deliverthe gradient solutions to the tube, care was taken to allow the fluid toflow slowly down the side of the tube at a 45° angle to insure that aproper interface formed between each layer of the gradient. The stepgradients, loaded with cells, were then centrifuged at 800×g for 20minutes without brake. After centrifugation, the tubes were carefullyremoved so as not to disturb each interface. Five distinct cellfractions resulted (4 bands and a pellet) (B1-B4, +Pellet) (see FIG. 1A,left conical tube). Each fraction was collected using either a steriledisposable bulb pipette or a 5 ml pipette and characterizedphenotypically and functionally (See Example 10 of Presnell et al.WO/2010/056328). When rodent kidney cell suspensions are subjected tostep-gradient fractionation immediately after isolation, the fractionenriched for tubular cells (and containing some cells from thecollecting duct) segments to a density between 1.062-1.088 g/mL. Incontrast, when density gradient separation was performed after ex vivoculture, the fraction enriched for tubular cells (and containing somecells from the collecting duct) segmented to a density between1.051-1.062 g/mL. Similarly, when rodent kidney cell suspensions aresubjected to step-gradient fractionation immediately after isolation,the fraction enriched for epo-producing cells, glomerular podocytes, andvascular cells (“B4”) segregates at a density between 1.025-1.035 g/mL.In contrast, when density gradient separation was performed after exvivo culture, the fraction enriched for epo-producing cells, glomerularpodocytes, and vascular cells (“B4”) segregated at a density between1.073-1.091 g/mL. Importantly, the post-culture distribution of cellsinto both the “B2” and the “B4” fractions was enhanced by exposure (fora period of about 1 hour to a period of about 24 hours) of the culturesto a hypoxic culture environment (hypoxia being defined as <21%(atmospheric) oxygen levels prior to harvest and step-gradientprocedures (additional details regarding hypoxia-effects on banddistribution are provided in Example 3).

Each band was washed by diluting with 3× the volume of KSFM, mixed well,and centrifuged for 5 minutes at 300×g. Pellets were re-suspended in 2mls of KSFM and viable cells were counted using trypan blue exclusionand a hemacytometer. 1 million cells were collected for an RNA sample,washed in PBS, and snap frozen in liquid nitrogen. The cells from B2 andB4 were used for transplantation studies into uremic and anemic femalerats, generated via a two-step 5/6 nephrectomy procedure at CharlesRiver Laboratories. Characteristics of B4 were confirmed by quantitativereal-time PCR, including oxygen-regulated expression of erythropoietinand vEGF, expression of glomerular markers (nephrin, podocin), andexpression of vascular markers (PECAM). Phenotype of the ‘B2’ fractionwas confirmed via expression of E-Cadherin, N-Cadherin, and Aquaporin-2.See FIGS. 49A and 49B of Presnell et al. WO/2010/056328.

Thus, use of the step gradient strategy allows not only the enrichmentfor a rare population of epo-producing cells (B4), but also a means togenerate relatively enriched fractions of functional tubular cells (B2)(see FIGS. 50 & 51 of Presnell et al. WO/2010/056328). The step gradientstrategy also allows EPO-producing and tubular cells to be separatedfrom red blood cells, cellular debris, and other potentially undesirablecell types, such as large cell aggregates and certain types of immunecells.

The step gradient procedure may require tuning with regard to specificdensities employed to provide good separation of cellular components.The preferred approach to tuning the gradient involves 1) running acontinuous density gradient where from a high density at the bottom ofthe gradient (16-21% Optiprep, for example) to a relatively low densityat the top of the gradient (5-10%, for example). Continuous gradientscan be prepared with any standard density gradient solution (Ficoll,Percoll, Sucrose, iodixanol) according to standard methods (AxisShield). Cells of interest are loaded onto the continuous gradient andcentrifuged at 800×G for 20 minutes without brake. Cells of similar sizeand granularity tend to segregate together in the gradients, such thatthe relative position in the gradient can be measured, and the specificgravity of the solution at that position also measured. Thus,subsequently, a defined step gradient can be derived that focusesisolation of particular cell populations based on their ability totransverse the density gradient under specific conditions. Suchoptimization may need to be employed when isolating cells from unhealthyvs. healthy tissue, or when isolating specific cells from differentspecies. For example, optimization was conducted on both canine andhuman renal cell cultures, to insure that the specific B2 and B4subpopulations that were identified in the rat were isolatable from theother species. The optimal gradient for isolation of rodent B2 and B4subpopulations consists of (w/v) of 7%, 11%, 13%, and 16% Optiprep. Theoptimal gradient for isolation of canine B2 and B4 subpopulationsconsists of (w/v) of 7%, 10%, 11%, and 16% Optiprep. The optimalgradient for isolation of human B2 and B4 subpopulations consists of(w/v) 7%, 9%, 11%, 16%. Thus, the density range for localization of B2and B4 from cultured rodent, canine, and human renal cells is providedin Table 2.1.

TABLE 2.1 Species Density Ranges. Step Gradient Species Density Rangesg/ml Band Rodent Canine Human B2 1.045-1.063 g/ml 1.045-1.058 g/ml1.045-1.052 g/ml B4 1.073-1.091 g/ml 1.063-1.091 g/ml 1.063-1.091 g/ml

Example 3—Low-Oxygen Culture Prior to Gradient Affects BandDistribution, Composition, and Gene Expression

To determine the effect of oxygen conditions on distribution andcomposition of prototypes B2 and B4, neokidney cell preparations fromdifferent species were exposed to different oxygen conditions prior tothe gradient step. A rodent neo-kidney augmentation (NKA) cellpreparation (RK069) was established using standard procedures for ratcell isolation and culture initiation, as described supra. All flaskswere cultured for 2-3 days in 21% (atmospheric) oxygen conditions. Mediawas changed and half of the flasks were then relocated to anoxygen-controlled incubator set to 2% oxygen, while the remaining flaskswere kept at the 21% oxygen conditions, for an additional 24 hours.Cells were then harvested from each set of conditions using standardenzymatic harvesting procedures described supra. Step gradients wereprepared according to standard procedures and the “normoxic” (21%oxygen) and “hypoxic” (2% oxygen) cultures were harvested separately andapplied side-by-side to identical step gradients. (FIG. 2). While 4bands and a pellet were generated in both conditions, the distributionof the cells throughout the gradient was different in 21% and 2%oxygen-cultured batches (Table 1). Specifically, the yield of B2 wasincreased with hypoxia, with a concomitant decrease in B3. Furthermore,the expression of B4-specific genes (such as erythropoietin) wasenhanced in the resulting gradient generated from the hypoxic-culturedcells (FIG. 73 of Presnell et al. WO/2010/056328).

A canine NKA cell preparation (DK008) was established using standardprocedures for dog cell isolation and culture (analogous to rodentisolation and culture procedures), as described supra. All flasks werecultured for 4 days in 21% (atmospheric) oxygen conditions, then asubset of flasks were transferred to hypoxia (2%) for 24 hours while asubset of the flasks were maintained at 21%. Subsequently, each set offlasks was harvested and subjected to identical step gradients (FIG. 3).Similar to the rat results (Example 1), the hypoxic-cultured dog cellsdistributed throughout the gradient differently than the atmosphericoxygen-cultured dog cells (Table 3.1). Again, the yield of B2 wasincreased with hypoxic exposure prior to gradient, along with aconcomitant decrease in distribution into B3.

TABLE 3.1 Rat (RK069) Dog (DK008) 2% O2 21% O2 2% O2 21% O2 B1  0.77% 0.24%  1.20%  0.70% B2 88.50% 79.90% 64.80% 36.70% B3 10.50% 19.80%29.10% 40.20% B4  0.23%  0.17%  4.40% 21.90%

The above data show that pre-gradient exposure to hypoxia enhancescomposition of B2 as well as the distribution of specific specializedcells (erythropoietin-producing cells, vascular cells, and glomerularcells) into B4. Thus, hypoxic culture, followed by density-gradientseparation as described supra, is an effective way to generate ‘B2’ and‘B4’ cell populations, across species.

Example 4—Isolation of Tubular/Glomerular Cells from Human Kidney

Tubular and glomerular cells were isolated and propagated from normalhuman kidney tissue by the enzymatic isolation methods describedthroughout. By the gradient method described above, the tubular cellfraction was enriched ex vivo and after culture. As shown in FIG. 68 ofof Presnell et al. WO/2010/056328 (incorporated herein by reference inits entirety), phenotypic attributes were maintained in isolation andpropagation. Tubular cell function, assessed via uptake of labeledalbumin, was also retained after repeated passage and cryopreservation.FIG. 69 of Presnell et al. WO/2010/056328 (incorporated herein byreference in its entirety) shows that when tubular-enriched andtubular-depleted populations were cultured in 3D dynamic culture, amarked increase in expression of tubular marker, cadherin, was expressedin the tubular-enriched population. This confirms that the enrichment oftubular cells can be maintained beyond the initial enrichment when thecells are cultured in a 3D dynamic environment.

Example 5—Further Separation of EPO-Producing Cells via Flow Cytometry

The same cultured population of kidney cells described above in Example2 was subjected to flow cytometric analysis to examine forward scatterand side scatter. The small, less granular EPO-producing cell populationwas discernable (8.15%) and was separated via positive selection of thesmall, less granular population using the sorting capability of a flowcytometer (see FIG. 70 of Presnell et al. WO/2010/056328 (incorporatedherein by reference in its entirety)).

Example 6—Characterization of an Unfractionated Mixture of Renal CellsIsolated from an Autoimmune Glomerulonephritis Patient Sample

An unfractionated mixture of renal cells was isolated, as describedabove, from an autoimmune glomerulonephritis patient sample. Todetermine the unbiased genotypic composition of specific subpopulationsof renal cells isolated and expanded from kidney tissue, quantitativereal time PCR (qrtper) analysis (Brunskill et al., supra 2008) wasemployed to identify differential cell-type-specific andpathway-specific gene expression patterns among the cell subfractions.As shown in Table 6.1, HK20 is an autoimmune glomerulonephritis patientsample. Table 6.2 shows that cells generated from HK20 are lackingglomerular cells, as determined by qRTPCR.

Example 7—Genetic Profiling of Therapeutically Relevant Renal BioactiveCell Populations Isolated from a Case of Focal SegmentalGlomerulosclerosis

To determine the unbiased genotypic composition of specificsubpopulations of renal cells isolated and expanded from kidney tissue,quantitative real time PCR (qrtper) analysis (Brunskill et al., supra2008) was employed to identify differential cell-type-specific andpathway-specific gene expression patterns among the cell subfractions.Human preparation HK023, derived from a case of focal segmentalglomerulosclerosis (FSGS) in which a large portion of glomeruli had beendestroyed, was evaluated for presence of glomerular cells in the B4fraction at the time of harvest. In brief, unfractionated (UNFX)cultures were generated (Aboushwareb et al., supra 2008) and maintainedindependently from each of (4) core biopsies taken from the kidney usingstandard biopsy procedures. After (2) passages of UNFX ex vivo, cellswere harvested and subjected to density gradient methods (as in Example8) to generate subfractions, including subfraction B4, which is known tobe enriched for endocrine, vascular, and glomerular cells based on workconducted in rodent, dog, and other human specimens.

TABLE 6.1 Cause of Death (D) Creatinine Etiology of or Kidney RemovalBUN sCreat Clearance HCT NB sPHOS Key Histopathologic Sample ID SpeciesAge/Gender Renal Disease (KR) (mg/dL) (mg/dL) (CC)/GFR/eGFR (%) (mg/dL)(mg/DL) uPRO Features PK001 Swine >1 yr/M Idiopathic (D) Renal 75 9.5 na34.1 10.6 6.3 na marked fibrosis; nephropathy Failure glomerularhypertrophy with focal sclerosis; tubular dilatation with protein castsPK002 Swine >1 yr/M no renal disease (D) na na na na na na na normalkidney Sacrifice histology DK001 Canine >11 yr/M age-related renal (D)24 1/1 ma 40.1 13.5 6.6 0 diffuse degeneration Sacrifice glomerular withfatty lipidosis with metaplasia of focal segmental flomeruli glomerularsclerosis DK002 Canine >2 yr/M chronic (D) 20 0.8 na 47 15.9 3.6 >3.0chronic glomerulonephritis Sacrifice glomerulonephritis with chronicinflammation, glomerular sclerosis, and moderate fibrosis HK016 Human 2mo/F no renal disease (D) Head 13 0.4 na 26.6 9.6 8.6 trace normalneonatal Trauma kidney histology HK017 Human 35 yr/F Petechial (D) CVA12 2.9 na 26 8.8 6.3 trace normal tubular hemorrhage histology; nosecondary to DIC fibrosis; fibrin thrombi throughout glomerularcapillaries HK018 Human 48 yr/F secondary to (D) CV/Renal 40 8.6 8.06(CC) 24.6 8.1 6.7 na marked fibrosis; hypertension, Failure (anuric)glomerular NIDDM, and sclerosis; tubular heart disease dilatation withprotein casts HK019 Human 52 yr/F secondary to (D) CV/Renal 127 5.7 14.5(CC) 23.7 8.4 12.4 >300 diffuse moderate hypertension, Failureglomerular NIDDM, and obsolescence heart disease with thickening ofBowman's capsule; peri- glomerulas fibrosis; moderate tubular injurywith diffuse tubulo-interstitial fibrosis, tubular dilatation withprotein casts. HK020 Human 54 yr/F auto-immune (D) CV/Stroke 94 16.64.35 (CC) 29 9.6 5.4 na Severe end- glomerulonephritis (anuric) stagerenal disease; no functional glomeruli observed; severe glomerularsclerosis and interstitial fibrosis with chronic inflammation, tubularcongestion with protein casts. HK021 Human 15 mo/M no renal disease (D)trauma 11 0.4 73.4 (CC) 29 10.3 3.4 trace normal kidney histology HK022Human 60 yr/M secondary to (D) CVA/ 53 3.3   17 (GFR) 31.1 10 1.8 100Severe end- hypertension Intracranial stage renal NIDDM, and hemorrhagedisease; diffuse heart disease severe glomerulosclerosis; interstitialfibrosis and tubular atrophy with protein casts. HK023 Human 18 yr/Mfocal segmental (KR) failed 28 6.4 13.8 (GFR) 36 11.8 6.4 na focalsegmental glomerulosclerosis, kidneys glomerulosclerosis nephroticremoved (10-15% of syndrome, prior to glomerufisclerosed), hypertensiontransplant associated with diffuse mesangial hypercellularity; diffuse,focally accentuated moderate to marked interstitial fibrosis and tubularatrophy; marked chronic active interstitial nephritis CKD Rats Rat 4-6mo/F renal mass (D) Renal 96.5 ± 14*  2.4 ± 0.2*  0.48 0.48 ± 0.3* 39.3± 1.8* 13.2 ± 0.6* 10.2 ± 1.2* 1420 ± 535* interstitial (5/6Nx) (Lewis)insufficiency Failure (eGFR) fibrosis; n = 16 glomerular atrophy andsclerosis; tubular degeneration and dilatation Healthy Rat 4-6 mo/F None(D) 16.9 ± 0.6* 0.4 ± 0.02* 1.7 ± 0.1* 46.1 ± 0.6* 14.7 ± 0.3*  6.8 ±0.3*  36 ± 13* normal adult rats (age- (Lewis) Sacrifice (eGFR) kidneyhistology matched; n = 16) Diabetic Rat 9 mo/M obesity, diabetes (D)30.9 ± 4.8* 0.6 ± 0.5*  3.8 ± 0.3* na na  5.3 ± 0.4*  931 ± 0.4*arteriolar Nephropathy (ZSF1) Sacrifice (eGFR) thickening, Rats severetubular (Ob/ObZSF1); degeneration, n = 10 dilation, and atrophy, andprotein casts in the Bowman's space and tubular lumens (REF: Prabhakar,2007 jASN); by 20 weeks of age Lean ZSF1 Rat 9 mo/M None (D) 18.9 ± 2.9*0.4 ± 0.05* 6.4 ± 1.2* na na  4.6 ± 0.5* 296 ± 69* moderate Rats (Age-(ZSF1) Sacrifice (eGFR) arteriolar Matched); thickening; n = 10 normaltubular and glomerular structures (REF: Prabhakar 2007 JASN); at 20weeks of age

TABLE 6.2 Compartmental analysis of cultured human, swine, and rat renalcells. Sample TUBULAR GLOMERULAR DUCTULAR OTHER ID E-CAD N-CAD AQP-1 CUBCYP24 ALB-U NEPH PODO AQP-2 EPO vEGF KDR CD31 SSC/FSC PK001 + nd nd ndnd ++ nd nd nd +R + nd nd + PK002 + nd nd nd nd + nd nd nd +R + nd nd +HK016 3.03 0.83 0.0001 0.0006 0.055 + 0.0004 0.0050 0.0001 0.020R 0.850.001 trace + HK017 0.66 0.83 0.0009 0.0002 0.046 ++ trace 0.0001 0.00030.032R 0.36 0.002 0.0003 + HK018 0.61 1.59 0.0001 0.0003 0.059 + 0.0002− − 0.004R 0.36 0.003 trace + HK019 0.62 2.19 0.026  0.0008 0.068 +/−0.0009 0.0003 0.0020 0.076R 0.40 0.002 0.0040 + HK020 0.07 1.65 0.00030.0007 0.060 +++ − − − 0.011R 0.40 0.002 − + Healthy + + + + + + + + ++R + + + + Lewis Rat (male) Rat CKD + + + + nd nd + + nd +R nd nd nd +model (5/6 NX Lewis)

The B4 fractions were collected separately from each independent UNFXsample of HK023, appearing as distinct bands of cells with buoyantdensity between 1.063-1.091 g/mL. RNA was isolated from each sample andexamined for expression of Podocin (glomerular cell marker) and PECAM(endothelial cell marker) by quantitative real-time PCR. As expectedfrom a biopsy-generated sample from a case of severe FSGS, the presenceof podocin(+) glomerular cells in B4 fractions was inconsistent, withpodocin undetectable in 2/4 of the samples. In contrast, PECAM+ vascularcells were consistently present in the B4 fractions of 4/4 of thebiopsy-initiated cultures. Thus, the B4 fraction can be isolated at the1.063-1.091 g/mL density range, even from human kidneys with severedisease states.

TABLE 7.1 Expression of Podocin and PECAM for detection of glomerularand vascular cells in subfraction B4 isolated from a case of FSGS.HK023/ RQ RQ Biopsy (Podocin)/B4 (PECAM)/B4 #1/p2 0.188 0.003 #2/p2 ND0.02 #3/p2 40.1 0.001 #4/p2 ND 0.003

Further, as shown in Table 7.2, human sample (HK018) displayedundetected Podocin (glomerular marker) by qRTPCR after density gradientcentrifugation.

TABLE 7.2 HK018 Post-Gradient gene expression characterization of B2 &B4’ Gene RQ(Unfx) RQ(B2) RQ(B4) B2/B4 Podocin 1 ND ND — VegF 1 1.43 1.620.9 Aqp1 1 1.7 1.2 1.4 Epo 1 0.9 0.5 1.8 Cubilin 1 1.2 0.7 1.7 Cyp 1 1.21.4 0.85 Ecad 1 1.15 0.5 2.3 Ncad 1 1.02 0.72 1.4

Example 8—Enrichment/Depletion of Viable Kidney Cell Types UsingFluorescent Activated Cell Sorting (FACS)

One or more isolated kidney cells may be enriched, and/or one or morespecific kidney cell types may be depleted from isolated primary kidneytissue using fluorescent activated cell sorting (FACS).

REAGENTS: 70% ethanol; Wash buffer (PBS); 50:50 Kidney cell medium (50%DMEM high glucose): 50% Keratinocyte-SFM; Trypan Blue 0.4%; Primaryantibodies to target kidney cell population such as CD31 for kidneyendothelial cells and Nephrin for kidney glomerular cells. Matchedisotype specific fluorescent secondary antibodies; Staining buffer(0.05% BSA in PBS) PROCEDURE: Following standard procedures for cleaningthe biological safety cabinet (BSC), a single cell suspension of kidneycells from either primary isolation or cultured cells may be obtainedfrom a T500 T/C treated flask and resuspend in kidney cell medium andplace on ice. Cell count and viability is then determined using trypanblue exclusion method. For kidney cell enrichment/depletion of, forexample, glomerular cells or endothelial cells from a heterogeneouspopulation, between 10 and 50e6 live cells with a viability of at least70% are obtained. The heterogeneous population of kidney cells is thenstained with primary antibody specific for target cell type at astarting concentration of 1 μg/0.1 ml of staining buffer/1×10⁶ cells(titer if necessary). Target antibody can be conjugated such as CD31 PE(specific for kidney endothelial cells) or un-conjugated such as Nephrin(specific for kidney glomerular cells).

Cells are then stained for 30 minutes on ice or at 4° C. protected fromlight. After 30 minutes of incubation, cells are washed bycentrifugation at 300×g for 5 min. The pellet is then resuspended ineither PBS or staining buffer depending on whether a conjugated isotypespecific secondary antibody is required. If cells are labeled with afluorochrome conjugated primary antibody, cells are resuspended in 2 mlsof PBS per 10e7 cells and proceed to FACS aria or equivalent cellsorter. If cells are not labeled with a fluorochrome conjugatedantibody, then cells are labeled with an isotype specific fluorochromeconjugated secondary antibody at a starting concentration of 1 ug/0.1ml/1e6 cells.

Cells are then stained for 30 min. on ice or at 4° C. protected fromlight. After 30 minutes of incubation, cells are washed bycentrifugation at 300×g for 5 min. After centrifugation, the pellet isresuspended in PBS at a concentration of 5e6/ml of PBS and then 4 mlsper 12×75 mm is transferred to a sterile tube.

FACs Aria is prepared for live cell sterile sorting per manufacturer'sinstructions (BD FACs Aria User Manual). The sample tube is loaded intothe FACs Aria and PMT voltages are adjusted after acquisition begins.The gates are drawn to select kidney specific cells types usingfluorescent intensity using a specific wavelength. Another gate is drawnto select the negative population. Once the desired gates have beendrawn to encapsulate the positive target population and the negativepopulation, the cells are sorted using manufacturer's instructions.

The positive target population is collected in one 15 ml conical tubeand the negative population in another 15 ml conical tube filled with 1ml of kidney cell medium. After collection, a sample from each tube isanalyzed by flow cytometry to determine purity. Collected cells arewashed by centrifugation at 300×g for 5 min. and the pellet isresuspended in kidney cell medium for further analysis andexperimentation.

Example 9—Enrichment/Depletion of Kidney Cell Types Using Magnetic CellSorting

One or more isolated kidney cells may be enriched and/or one or morespecific kidney cell types may be depleted from isolated primary kidneytissue.

REAGENTS: 70% ethanol, Wash buffer (PBS), 50:50 Kidney cell medium (50%DMEM high glucose): 50% Keratinocyte-SFM, Trypan Blue 0.4%, RunningBuffer(PBS, 2 mM EDTA,0.5% BSA), Rinsing Buffer (PBS,2 mM EDTA),Cleaning Solution (70% v/v ethanol), Miltenyi FCR Blocking reagent,Miltenyi microbeads specific for either IgG isotype, target antibodysuch as CD31(PECAM) or Nephrin, or secondary antibody.

PROCEDURE: Following standard procedures for cleaning the biologicalsafety cabinet (BSC), a single cell suspension of kidney cells fromeither primary isolation or culture is obtained and resuspended inkidney cell medium. Cell count and viability is determined using trypanblue exclusion method.

For kidney cell enrichment/depletion of, for example, glomerular cellsor endothelial cells from a heterogeneous population, at least 10e6 upto 4e9 live cells with a viability of at least 70% is obtained.

The best separation for enrichment/depletion approach is determinedbased on target cell of interest. For enrichment of a target frequencyof less than 10%, for example, glomerular cells using Nephrin antibody,the Miltenyi autoMACS, or equivalent, instrument program POSSELDS(double positive selection in sensitive mode) is used. For depletion ofa target frequency of greater than 10%, the Miltenyi autoMACS, orequivalent, instrument program DEPLETES (depletion in sensitive mode) isused.

Live cells are labeled with target specific primary antibody, forexample, Nephrin rb polyclonal antibody for glomerular cells, by adding1 μg/10e6 cells/0.1 ml of PBS with 0.05% BSA in a 15 ml conicalcentrifuge tube, followed by incubation for 15 minutes at 4° C.

After labeling, cells are washed to remove unbound primary antibody byadding 1-2 ml of buffer per 10e7 cells followed by centrifugation at300×g for 5 min. After washing, isotype specific secondary antibody,such as chicken anti-rabbit PE at 1 ug/10e6/0.1 ml of PBS with 0.05%BSA, is added, followed by incubation for 15 minutes at 4° C.

After incubation, cells are washed to remove unbound secondary antibodyby adding 1-2 ml of buffer per 10e7 cells followed by centrifugation at300×g for 5 min. The supernatant is removed, and the cell pellet isresuspended in 60 μl of buffer per 10e7 total cells followed by additionof 20 μl of FCR blocking reagent per 10e7 total cells, which is thenmixed well. Add 20 μl of direct MACS microbeads (such as anti-PEmicrobeads) and mix and then incubate for 15 min at 4° C.

After incubation, cells are washed by adding 10-20× the labeling volumeof buffer and centrifuging the cell suspension at 300×g for 5 min. andresuspending the cell pellet in 500 μl-2 mls of buffer per 10e8 cells.

Per manufacturer's instructions, the autoMACS system is cleaned andprimed in preparation for magnetic cell separation using autoMACS. Newsterile collection tubes are placed under the outlet ports. The autoMACScell separation program is chosen. For selection the POSSELDS program ischosen. For depletion the DEPLETES program is chosen.

The labeled cells are inserted at uptake port, then beginning theprogram. After cell selection or depletion, samples are collected andplaced on ice until use. Purity of the depleted or selected sample isverified by flow cytometry.

Example 10—Cells with Therapeutic Potential can be Isolated andPropagated from Normal and Chronically-Diseased Kidney Tissue

The objective of the present study was to determine the functionalcharacterization of human NKA cells through high content analysis (HCA).High-content imaging (HCI) provides simultaneous imaging of multiplesub-cellular events using two or more fluorescent probes (multiplexing)across a number of samples. High-content Analysis (HCA) providessimultaneous quantitative measurement of multiple cellular parameterscaptured in High-Content Images. In brief, unfractionated (UNFX)cultures were generated (Aboushwareb et al., supra 2008) and maintainedindependently from core biopsies taken from five human kidneys withadvanced chronic kidney disease (CKD) and three non-CKD human kidneysusing standard biopsy procedures. After (2) passages of UNFX ex vivo,cells were harvested and subjected to density gradient methods (as inExample 2) to generate subfractions, including subfractions B2, B3,and/or B4.

Human kidney tissues were procured from non-CKD and CKD human donors assummarized in Table 10.1. FIG. 4 shows histopathologic features of theHK17 and HK19 samples. Ex vivo cultures were established from allnon-CKD (3/3) and CKD (5/5) kidneys. High content analysis (HCA) ofalbumin transport in human NKA cells defining regions of interest (ROI)is shown in FIG. 5 (HCA of albumin transport in human NKA cells).Quantitative comparison of albumin transport in NKA cells derived fromnon-CKD and CKD kidney is shown in FIG. 6. As shown in FIG. 6, albumintransport is not compromised in CKD-derived NKA cultures. Comparativeanalysis of marker expression between tubular-enriched B2 and tubularcell-depleted B4 subfractions is shown in FIG. 7 (CK8/18/19).Table 10.1

TABLE 10.1 Cause of Creatinine Death (D) Clearance Etiology or KidneyBUN (CC)/ HB sPHOS Sample Age/ of Renal Removal (mg/ sCREAT GFR/ HCT(mg/ (mg/ Key Histopathologic ID Gender Disease (KR) dL) (mg/dL) eGFR(%) dL) dL) uPRO Features HK016  2 mo/F no renal (D) Trauma 13 0.4 na26.6 9.6 8.6 trace normal neonatal kidney disease histology HK017 35yr/F Petechial (D) CVA 12 2.9 na 26 8.8 6.3 trace normal tubularhistology; no hemorrhage fibrosis; fibrin thrombi secondary throughoutglomerular to DIC capillaries HK018 43 yr/F secondary to (D) CV/ 40 8.68.06 (CC) 24.6 8.1 6.7 na marked fibrosis; glomerular hypertension,Renal (anuric) sclerosis; tubular dilatation NIDDM, Failure with proteincasts and heart disease HK019 52 yr/F secondary to (D) CV/ 127 5.7 14.5(CC) 23.7 8.4 12.4 >300 diffuse moderate glomerular hypertension, Renalobsolescence with thickening NIDDM, Failure of Bowman's capsule; peri-and heart glomerular fibrosis; moderate disease tubular injury withdiffuse tubulo-interstitial fibrosis, tubular dilatation with proteincasts. HK020 54 yr/F auto-immune (D) CV/ 94 16.6 4.35 (CC) 29 9.6 5.4 naSevere end-stage renal glomerulone- Stroke (anuric) disease; nofunctional phritis glorneruli observed; severe glomerular sclerosis andinterstitial fibrosis with chronic inflammation; tubular congestion withprotein casts. HK021 15 mo/M no renal (D) Trauma 11 0.4 73.4 (CC) 2910.3 3.4 trace normal kidney histology disease HK022 60 yr/M secondaryto (D) CVA/ 53 3.3 17 31.1 10 1.8 100 Severe end-stage renalhypertension, Intracranial (GFR) disease; diffuse severe NIDDMhemorrhage glomerulosclerosis; and heart interstitial fibrosis andtubular disease atrophy with protein casts HK023 18 yr/M focal (KR)failed 28 6.4 13.8 (GFR) 36 11.8 6.4 na focal segmental segmentalkidneys glomerulosclerosis (10-15% glomerulo- removed of glomerulisclerosed), sclerosis, prior to associated with diffuse nephrotictransplant mesangial hypercellularity; syndrome, diffuse, focallyaccentuated hypertension moderate to marked interstitial fibrosis andtubular atrophy; marked chronic active interstitial nephritis

Comparative functional analysis of albumin transport betweentubular-enriched B2 and tubular cell-depleted B4 subfractions is shownin FIG. 8. Subfraction B2 is enriched in proximal tubule cells and thusexhibits increased albumin-transport function.

Albumin uptake: Culture media of cells grown to confluency in 24-well,collagen IV plates (BD Biocoat™) was replaced for 18-24 hours withphenol red-free, serum-free, low-glucose DMEM (pr-/s-/lg DMEM)containing 1X antimycotic/antibiotic and 2 mM glutamine. Immediatelyprior to assay, cells were washed and incubated for 30 minutes withpr-/s-/lg DMEM+10 mM HEPES, 2 mM glutamine,1.8 mM CaCl2, and 1 mM MgCl2.Cells were exposed to 25 μg/mL rhodamine-conjugated bovine albumin(Invitrogen) for 30 min, washed with ice cold PBS to stop endocytosisand fixed immediately with 2% paraformaldehyde containing 25 μg/mLHoechst nuclear dye. For inhibition experiments, 1 μMreceptor-associated protein (RAP) (Ray Biotech, Inc., Norcross Ga.) wasadded 10 minutes prior to albumin addition. Microscopic imaging andanalysis was performed with a BD Pathway™ 855 High-Content Biolmager(Becton Dickinson) (see Kelley et al. Am J Physiol Renal Physiol. 2010November; 299(5):F1026-39. Epub Sep. 8, 2010).

In conclusion, HCA yields cellular level data and can reveal populationsdynamics that are undetectable by other assays, i.e., gene or proteinexpression. A quantifiable ex-vivo HCA assay for measuring albumintransport (HCA-AT) function can be utilized to characterize human renaltubular cells as components of human NKA prototypes. HCA-AT enabledcomparative evaluation of cellular function, showing that albumintransport-competent cells were retained in NKA cultures derived fromhuman CKD kidneys. It was also shown that specific subfractions of NKAcultures, B2 and B4, were distinct in phenotype and function, with B2representing a tubular cell-enriched fraction with enhanced albumintransport activity. The B2 cell subpopulation from human CKD arephenotypically and functionally analogous to rodent B2 cells thatdemonstrated efficacy in vivo (as shown above).

Example 11—Marker Expression as a Predictor of Renal Regeneration

This study concerns stem and progenitor marker expression as a predictorof renal regeneration in 5/6 nephrectomized rats treated withtherapeutically bio-active primary renal cell sub-populations. Theunderlying mechanisms by which NKA treatment improved renal function arebeing characterized. Our studies on NKA treatment's mechanism of actionconcern cell-cell signalling, engraftment, and fibrotic pathways. Thepresent work focused on how NKA treatment might increase the organ'sintrinsic regenerative capacity—perhaps by mobilizing renal stem cells.We hypothesize that the extended survival and improvement in renalfunction observed in NKA-treated 5/6 NX rats is associated withmolecular expression of specific stem cell markers.

Using a rat 5/6 nephrectomy model for CKD, this study employs molecularassays to evaluate the mobilization of resident stem and progenitorcells within the rat 5/6 nephrectomized kidney in response to directinjection with defined, therapeutically bio-active primary renal cellpopulations. It was observed that this cell-based therapy isspecifically associated with up-regulation of the key stem cell markersCD24, CD133, UTF1, SOX2, LEFTY1, and NODAL at both transcript andprotein levels. Up-regulation was detected by 1 week post-injection andpeaked by 12 weeks post-injection. Activation of stem and progenitorcell markers was associated with increased survival and significantimprovement of serum biomarkers relative to untreated nephrectomizedcontrols.

Materials and Methods

Isolation of primary renal cell populations from rat. Isolation ofprimary renal cell populations from rat were performed as previouslydescribed (Aboushwareb et al., supra 2008; Presnell et al., 2009 FASEB J23: LB143).

In vivo study design and analysis. Detailed descriptions of theisolation of primary renal cell populations (Presnell et al. Tissue EngPart C Methods. 2010 Oct. 27. [Epub ahead of print]) and the in vivostudies that evaluated the bioactivity of primary renal cellsub-populations in the 5/6 nephrectomized rodent model of CKD (Kelley etal. supra 2010). A tubular cell-enriched subpopulation of primary renalcells improves survival and augments kidney function in a rodent modelof chronic kidney disease were published elsewhere. In the currentstudy, tissues were isolated at necropsy from rats treated with B2 (NKA#1) or a B2+B4 mixture (NKA #2) and compared to nephrectomized (Nx) andsham-operated, non-nephrectomized rats (Control). In FIGS. 9 and 11 andTable 11.1, data from NKA #1 and NKA #2 treated rats was pooled.Systemic data was obtained by analysis of blood samples drawn weekly andpre-necropsy from rats on study.

Table 11.1 shows survival data for sham treated animals (control), n=3;nx control (Nx), n+3; animals treated with B2 cells (NKA #1), n=7; B2+B4cells (NKA#2); n=7. At the end of the study (23-24 weeks) none of the Nxanimals remained. NKA treated animals had a superior survival ratecompared to the untreated Nx control.

TABLE 11.1 Treatment Early Midpoint End of Study Group 1 week 12-13 Week[23-24 Week] Control 2/3*  2/2  2/2  NX 2/3*  1/2  0/1  NKA #1 5/7**3/7** 1/3** NKA #2 5/7** 3/7** 3/3  *1 animal sacrificed at scheduledtimepoint for tissue **2 animals sacrificed at scheduled timepoint fortissue

RNA Isolation, cDNA Synthesis and qRT-PCR. RNA was isolated from tissuesembedded in optimum cutting temperature (OCT) freezing media as follows:tissue blocks were placed at room temperature and excess OCT wasremoved, the tissues were then placed in PBS to allow complete thawingand removal of residual OCT, the tissues were washed three times in PBSand then coarsely chopped and aliquoted into microfuge tubes. Thealiquoted tissues were then pulverized using a pestle and RNA wasextracted using the RNeasy Plus Mini Kit (Qiagen, Valencia Calif.). RNAintegrity was determined spectrophotomerically and cDNA was generatedfrom a volume of RNA equal to 1.4 μg using the SuperScript® VILO™ cDNASynthesis Kit (Invitrogen, Carlsbad Calif.). Following cDNA synthesis,each sample was diluted 1:6 by adding 200 μl of diH₂O to bring the finalvolume to 240 μl. The expression levels of target transcripts wereexamined via quantitative real-time PCR (qRT-PCR) using cataloguedprimers and probes from ABI and an ABI-Prism 7300 Real Time PCR System(Applied Biosystems, Foster City Calif.). Amplification was performedusing the TaqMan® Gene Expression Master Mix (ABI, Cat #4369016) andpeptidylprolyl isomerase B (PPIB) was utilized as the endogenouscontrol. qRT-PCR Reaction: 10 μl Master Mix (2X), 1 μl Primer and Probe(20X), 9 μl cDNA, 20 μl Total Volume per Reaction. Each reaction wassetup as follows using the ^(TaqMan)® primers and probes.

Gene Abbreviation TaqMan primer SRY (sex determining region Y)-box 2Sox2 Rn01286286_g1 Undifferentiated Embryonic Cell Transcription FactorUTF1 Rn01498190_g1 Nodal Homolog from Mouse NODAL Rn01433623_m1 Prominin1 CD133 Rn00572720_m1 CD24 CD24 Rn00562598_m1 LEFTY1

Western Blot. Frozen whole kidney tissue embedded in OCT freezing mediawas utilized for protein sample collection. OCT was removed as describedabove and all tissues were lysed in a buffer consisting of 50 mM Tris(pH 8.0), 120 mM NaCl, 0.5% NP40, and protease inhibitor cocktail (RocheApplied Science, Indianapolis Ind.). Lysis proceeded for 15 minutes atroom temperature with rocking followed by centrifugation for 10 minutesat 13,000 RPM. All supernatants were collected and proteinconcentrations were determined by Bradford Assay. SDS PAGE Gel wascarried out by adding 30 μg of protein per sample to each well ofNuPAGE® Novex 10% Bis-Tris Gels (Invitrogen). The gels wereelectrophoresed for 40 min at 200V in IVIES running buffer (Invitrogen).The proteins were then transferred to nitrocellulose membranes using theI-Blot system (Invitrogen), and blocked with 15 mL of 4% w/v low-fatmilk dissolved in Tris Buffered Saline with 0.1% Tween-20 (TBS-T)(Sigma, St. Louis, Mo.) for 2 hours at room temperature. The membraneswere probed overnight at room temperature with the following antibodies:each diluted in 5 mL TBS-T with 2% w/v low-fat milk. (Anti-Human

Lefty-A Long & Short isoforms (R&D systems MAB7461); Anti-Human, Mouse &Rat CD133 (Abcam AB19898); Anti-Human & Mouse UTF1 (Millipore MAB4337);Anti-Human NODAL (Abcam AB55676); Anti-Human & Rat CDH11 (OB Cadherin)(Thermo Scientific MA1-06306); Anti-Rat CD24 (Becton Dickinson)). Themembranes were washed 3 times/10 minutes each with TBS-T, then probedwith the appropriate HRP-conjugated secondary antibody (Vector LabsPI-2000; PI-1000) diluted in TBS-T with 2% w/v low-fat milk (1:60,000)for 1.5 hours at room temperature. The membranes were washed 3 times/10minutes each in TBS-T, followed by two 10-minute washes in diH₂O. Theblots were developed using ECL Advance chemiluminescent reagent (GEHealthcare Life Sciences, Piscataway N.J.) and visualized using theChemiDoc™ XRS molecular imager and Quantity One® software (BioRad,Hercules Calif.).

Results. Molecular assays to evaluate the mobilization of resident stemand progenitor cells in 5/6 NX rats were developed and used toinvestigate the temporal response of these markers to NKA treatment. Itwas observed that NKA treatment was specifically associated withup-regulation of the key stem cell markers CD24, CD133, UTF-1, SOX-2,LEFTY, and NODAL at mRNA transcript and protein levels. Up-regulationwas detected by 1 week post-injection and had peaked by 12 weekspost-injection. Activation of stem and progenitor cell markers wasassociated with increased survival and significant improvement of serumbiomarkers (i.e., improvement of renal filtration) relative to untreated5/6 NX control animals.

FIG. 9 shows the expression of SOX2 mRNA in host tissue after treatmentof 5/6 NX rats with NKA. Temporal analysis of SOX2 mRNA expressionshowed 1.8-fold increase in SOX2 mRNA within NKA treatment group over Nxcontrol by 12 week post-implantation. A 2.7-fold increase in SOX2 mRNAexpression was observed in NKA treatment group over Nx control by 24weeks post-implantation. (1-week n=3 each for Control (sham), Nx(control), and NKA treated) (12 week n=1 each for Control (sham) and Nx(control); NKA treated n=4) (24 week n=1 each for Control (sham) and Nx(control); NKA treated n=4). *Indicates p-value=0.023 or <0.05.

FIG. 10—Western blot showing time course of expression of CD24, CD133,UTF1, SOX2, NODAL and LEFTY in sham control (Control), Nx control (Nx),and rats treated NKA #1 and NKA #2 at 1, 12 and 24 weeks post-treatment.Frozen whole kidney tissue (N=1 for each sample) embedded in OCTfreezing media was utilized for protein sample collection. Lanes werenormalized by total mass protein loaded. CD133, UTF1, NODAL, LEFTY andSOX2 protein levels in NKA-treated tissues were elevated relative toControl or Nx rats at all time points.

FIG. 11 depicts a time course of regenerative response index (RRI). Adensitometric analysis of individual protein expression (FIG. 10) wasused to generate a quantitative index of regenerative marker proteinexpression, or regenerative response index (RRI). Band intensity wascalculated from each western blot using Image J v1.4 software (NIH) andvalues normalized per unit area for each protein. Average intensity wasdetermined for sham, Nx, and NKA treatment groups by compiling the 5markers used in the western blot analysis for each time point. Plotshows XY scatter with smoothed line fit generated from 1, 12,and 24 weektime points. The average intensity for each group was plotted over timeto highlight the trends in the host tissue response of stem cell markerprotein expression. Statistical analysis was performed using standardtwo tailed Student's t-test assuming equal variance for each sample.Confidence interval of 95% (p-value<0.05) was used to determinestatistical significance. (NKA treated group n=2; Control (sham) n=1; Nx(control) n=1). In sham control animals, RRI shows only a slightreduction from 90.47 at 1 week post-treatment to 81.89 at 24 weeks posttreatment. In contrast, kidney from 5/6 Nx controls presents essentiallythe opposite response, with RRI increasing from 82.26 at 1 weekpost-treatment to 140.56 at 18 weeks post-treatment, at which point theanimal died. In NKA-treated animals RRI increased sharply from 62.89 at1 week post-treatment to 135.61 by 12 weeks post-treatment and fell to112.61 by 24 weeks post-treatment.

NKA treatment was observed to be associated with up-regulation of thestem cell markers CD24, CD133, UTF-1, SOX-2 and NODAL at both transcriptand protein levels in the host tissue. Up-regulation was detected by 1week post-treatment and peaked by 12 weeks post-treatment. Overallactivation of stem and progenitor cell markers in host tissues wasassociated with increased survival (1) and improvement ofclinically-relevant serum biomarkers relative to untreatednephrectomized controls.

Mobilization of resident stem and progenitor cell populations inresponse to NKA treatment may contribute to the restoration of kidneyfunction in 5/6 NX animals by regenerating damaged kidney tissue andorgan architecture. The molecular assays used in this study mighttherefore provide a rapid, straightforward, and predictive assay ofregenerative outcomes for evaluating tissue engineering and regenerativemedicine treatments for CKD.

Example 12—Exosomes Derived from Primary Renal Cells Contain MicroRNAs

We sought to correlate specific exosome-derived miRNAs withfunctionally-relevant outcomes in target cells in vitro to inform thedesign of in vivo studies for elucidating mechanisms that yieldregenerative outcomes.

METHODS: The effect of conditioned media on signaling pathwaysassociated with regenerative healing responses was investigated usingcommercially available cells: HK-2 (human proximal tubule cell line),primary human renal mesangial cells (HRMC), and human umbilical cordendothelial cells (HUVEC). RNA content from exosomes in conditionedmedia from human and rat primary renal cell cultures (UNFX) was screenedby PCR-based array designed to detect known miRNAs. Low oxygen has beenreported to affect exosome shedding; therefore, a group of cultures wasexposed to low oxygen (2% O₂) for 24 hours prior to media collection.Exosomes were separated from cellular debris by FACS.

FIG. 12 provides a schematic for the preparation and analysis of UNFXconditioned media.

RESULTS: UNFX-conditioned media was found to affect signaling pathwaysassociated with regenerative healing responses; these responses were notobserved in controls using non-conditioned media. Specifically, NFKB(immune response) and epithelial-to-mesenchymal transition (fibroticresponse) was attenuated in HK-2 cells, PAI-1 (fibrotic response) wasattenuated in HRMC cells, and angiogenesis was promoted in HUVEC.Preliminary data from PCR array screening of exosome content fromUNFX-conditioned media indicates that UNFX produces exosomes containingmiRNA sequences consistent with the observed responses toUNFX-conditioned media.

FIGS. 13A-C show that conditioned media from UNFX cultures affectsmultiple cellular processes in vitro that are potentially associatedwith regenerative outcomes. NFkB signaling is proposed as a key mediatorof inflammatory processes in kidney diseases (Rangan et al., 2009. FrontBiosci 12:3496-3522; Sanz et al., 2010. J Am Soc Nephrol 21:1254-1262),and can be activated by Tumor Necrosis Factors (TNF). HK-2 cells werepreincubated with unconditioned media (left) or UNFX conditioned media(right) for 1 hour at 37° C., then activated with or without 10 ng/mlTNFa.

FIG. 13A shows that UNFX-conditioned media attenuates TNF-a mediatedactivation of NF-kB. NFkB activation was measured by RelA/p65immunofluorescence staining (green). Hoechst-counter-stained nuclei(blue) and phalloidin-stained filamentous actin (red) facilitateassessment of RelA/p65 nuclear localization (white arrows).

FIG. 13B shows that UNFX-conditioned media increases proangiogenicbehavior of HUVEC cell cultures. HUVEC cells (100,000 per well) wereoverlaid onto polymerized Matrigel in Media 200 plus 0.5% BSA.Unconditioned media (left) or UNFX-conditioned medium (right) was addedand cellular organizational response was monitored visually for 3-6hours with image capture. Cellular organization was scored for cellmigration (white arrowheads), alignment (black arrowheads), tubuleformation (red arrowheads), and formation of closed polygons(asterisks). UNFX conditioned media induced more tubules and closedpolygons compared to unconditioned media, suggesting that proangiogenicfactors are present in the media.

FIG. 13C shows that UNFX-conditioned media attenuates fibrosis pathwaysin epithelial cells. HK-2 cells lose epithelial characteristics, andacquire a mesenchymal phenotype when exposed to Transforming GrowthFactors (TGF) in vitro, replicating the epithelial-to-mesenchymaltransition (EMT) that is associated with progression of renal fibrosis(Zeisberg et al. 2003 Nat Med 9:964-968). HK-2 cells were cultured inunconditioned media (CTRL), unconditioned media containing 10 ng/mlTGFβ1 (TGFβ1), or UNFX conditioned media containing 10 ng/ml TGFβ1(TGFβ1+CM) for 72 hours. Cells were assayed by quantitative RT-PCR forCDH1 (epithelial marker), CNN1 (mesenchymal marker) and MYH11(mesenchymal marker). Conditioned media reduces the degree ofTGFβ1-induced EMT as measured by CDH1, CNN1, and MYH11 gene expression.Error bars represent the stardard error of the mean (SEM) of threeexperimental replicates.

FIG. 13D depicts the positive feedback loop established by TGFβ1 andPlasminogen Activator Inhibitor-1 (PAI-1) that, when left unchecked, canlead to the progressive accumulation of extracellular matrix proteins(Seo et al., 2009. Am J Nephrol 30:481-490).

FIGS. 14A-B show the attenuation of fibrosis pathways in mesangialcells. HRMC were cultured for 24 hours in control (CTRL) or UNFXconditioned media (UNFX CM) with (+) or without (−) the addition of 5ng/ml TGFβ1. Western blot analysis for PAI-1 demonstrates that UNFX CMattenuates the TGFβ1-induced increase in PAI-1 protein levels. bActin isshown as a loading control. Human renal mesangial cells (HRMC) expressincreased levels of PAI-1 in the presence (+) of 5 ng/ml TGFb1.Co-culture with conditioned media (CM) derived from human bioactivekidney cells attenuates TGFb1-induced PAI-1 protein expression. PAI-1expression at the mRNA level was unaltered by CM (data not shown).

FIG. 14B shows that CM from rat bioactive kidney cells had similareffect on cultured HRMC induced (+) and uninduced (−) with TGFb1. CMsupernatant (Deplete Rat CM) collected after centrifugation was lesseffective at attenuating PAI-1 expression, suggesting that the CMcomponent responsible for the observed attenuation of PAI-1 proteinmight be associated with vesicles secreted by the rat bioactive kidneycells.

FIGS. 15A-C show that the conditioned media from UNFX contains secretedvesicles. FIG. 15 depicts secreted vesicles (including exosomes), whichare bilipid structures (red) that encompass cytoplasm-derived internalcomponents (green). Phosphatidylserines (blue triangles) are componentsof the membrane that are exposed to the extracellular space duringvesicle biogenesis (Thery et al., 2010. Nat Rev Immunol 9:581-593).

PKH26 and CFSE label the lipid membrane and cytoplasm of secretedvesicles (Aliotta et al., 2010. Exp Hematol 38:233-245), respectively,while Annexin V binds phosphatidylserines.

FIGS. 15B-C shows FACS sorting. UNFX conditioned media was labeled withPKH26, CFSE, and APC-conjugated Annexin V, then sorted byfluorescence-assisted cell sorting (FACS). Triple-positive particles,representing secreted vesicles, were collected and total RNA wasextracted using TRIZol reagent. microRNA content was screened for knownsequences using commercially available RT-PCR-based arrays.

Table 12.1 shows that secreted vesicles contain microRNAs with predictedtherapeutic outcomes. UNFX cells shed exosomes that contain known miRNAsequences. UNFX-conditioned media affects functionally-relevantregenerative responses in human cell lines. The cause and effectrelationship between detected miRNAs and observed regenerative responsesis under active investigation; however, the results achieved to datesuggest that UNFX cells have the potential to producetherapeutically-relevant paracrine effects via exosome-mediated transferof miRNAs to target cells and tissues.

TABLE 12.1 miRNA in exosomes Gene targets Predicted effects miR-146aTRAF6, IRAK1* Inhibits NFkB miR-130a GAX, HOXA5** Promotes angiogenesismiR-23b Smad 3/4/5*** Inhibits TGFβ signal transduction (anti-fibrotic)*Taganov et al, 2006. Proc Natl Acad Sci USA 103:12481-12486. **Chen andGorski, 2008. Blood 111:1217-1226. ***Rogler et al., 2009. Hepatology50:575-584.The data support the conclusion that excreted vesicles from bioactiverenal cell cultures contain components that attenuate PAI-1 induced bythe TGFb1/PAI-1 feedback loop.

Microarray and RT-PCR analysis. Unfractionated (UNFX) bioactive renalcells from Lewis rats were cultured in basal media (50:50 mix of DMEMand KSFM without serum or supplements) for 24 hours under low oxygenconditions (2% O2). Conditioned media was collected and ultracentrifugedat 100,000×g for 2 hours at 4C to pellet secreted vesicles (e.g.microvesicles, exosomes). Total RNA was extracted from the resultingpellet, and assayed for known microRNA species by real time RT-PCR (RatMicroRNA Genome V2.0 PCR Array; Qiagen # MAR-100A). The following miRNAswere detectable.

miR-21 let-7b miR-26b miR-23a miR-20b-5p miR-200a miR-30c miR-29amiR-126 miR-1224 let-7d miR-29c miR-23b miR-22 miR-200c miR-92a miR-322miR-151 miR-100 let-7e miR-429 miR-125b-5p miR-191 miR-103 miR-195miR-99b let-7a miR-10a-5p miR-19b miR-322* miR-370 miR-10b miR-15bmiR-24 miR-27b miR-378 miR-30a miR-125a-5p miR-127 miR-16 miR-30dmiR-199a-5p miR-126* miR-31 miR-181b miR-30b-5p miR-93 miR-106b miR-27amiR-182 miR-196c miR-20a miR-99a miR-196b let-7c miR-320 miR-19a miR-26amiR-664 miR-145 miR-17-5p miR-30e* let-7f miR-30e let-7i miR-181d miR-25miR-196a miR-181a miR-221 miR-151* miR-22* miR-30a* miR-328 miR-96miR-351 miR-185 miR-34a miR-218 miR-28 miR-223 miR-210 miR-192 miR-301bmiR-98 miR-92b miR-505 miR-18a miR-672 miR-532-3p miR-342-3p miR-150miR-7a miR-203 miR-425 miR-451 miR-352 miR-146a miR-34c* miR-181cmiR-107 miR-339-3p miR-222 miR-330* miR-190 miR-219-1-3p miR-409-3pmiR-671 miR-708 miR-877 miR-465 miR-652 miR-760-3p miR-674-3p let-7d*miR-770 miR-21* miR-503 miR-152 miR-99b* miR-138 miR-106b* miR-125a-3pmiR-450a miR-675 miR-183 miR-365 miR-423 miR-143 miR-874 miR-194miR-324-5p miR-345-5p miR-490 miR-674-5p miR-374 miR-128 miR-760-5pmiR-872 miR-497 miR-361 miR-186 miR-301a miR-296 miR-130a miR-130bmiR-148b-3p miR-140* miR-199a-3p miR-542-3p miR-28* miR-326 miR-667miR-212 miR-132 miR-935 miR-139-3p miR-375 miR-24-2* miR-347 miR-25*miR-433 miR-295 miR-142-3p miR-138* miR-140 miR-7a* miR-190b miR-9miR-298 let-7i* miR-871 let-7b* miR-484 miR-29a* miR-338 miR-30c-2*miR-542-5p miR-449a miR-125b-3p miR-129 miR-125b* miR-764 miR-214miR-34c miR-421 miR-29c* miR-346 miR-485 miR-489 miR-147 miR-296*miR-141 miR-9* miR-29b miR-500 miR-146b miR-382 miR-17-3p miR-219-5pmiR-511 miR-339-5p miR-653 miR-99a* miR-7b miR-340-3p miR-499 miR-501miR-224 miR-101a* miR-206 miR-330 miR-20b-3p miR-193* miR-544 miR-434miR-350 miR-193 miR-139-5p miR-466b miR-761 miR-10a-3p miR-205 miR-181a*miR-148b-5p miR-598-5p miR-27a* miR-598-3p miR-532-5p miR-34b miR-29b-2*miR-411 miR-455 miR-335 miR-188 miR-20a* miR-215 miR-331 miR-488miR-219-2-3p miR-124 miR-26b* miR-327 miR-431 miR-342-5p miR-204 miR-154miR-543 miR-504 miR-30d* miR-105 let-7e* miR-466c miR-291a-5p miR-129*miR-101b miR-216a miR-201 miR-122 miR-496 miR-483 miR-539 miR-379miR-742 miR-30b-3p miR-711 miR-1 miR-184 miR-32 miR-293 miR-673miR-291a-3p miR-349 miR-463 miR-487b miR-30c-1* miR-297 miR-23a* miR-153miR-363* miR-294 miR-743a miR-384-5p miR-207 miR-323 miR-344-5p miR-343miR-495 miR-133a miR-292-3p miR-410 miR-582 miR-376a miR-137 miR-541miR-540 miR-300-3p miR-344-3p miR-336 miR-369-5p miR-345-3p miR-134miR-376b-3p miR-362 miR-24-1* miR-211 miR-380 miR-363 miR-33 miR-878miR-329 miR-758 miR-377 miR-133b miR-802 miR-409-5p miR-875

Example 13—Paracrine Factors Derived from Bioactive Kidney Cells

In the present study, we employed in vitro cell-based assays toinvestigate potential paracrine mechanism(s) by which bioactive kidneycells could modulate fibrosis through mediators such as PlasminogenActivator Inhibitor-1 (PAI-1).

Materials and Methods: Conditioned media was collected from rat andhuman cultures of bioactive kidney cells (Aboushwareb et al., World JUrol 26, 295, 2008; Presnell et al. 2010 supra) under serum- andsupplement-free conditions and utilized for in vitro assays.Commercially available rat- and human-derived mesangial cells were usedas surrogates for host-response tissues in the in vitro assays becausemesangial cells are a source of PAI-1 production in injured or diseasedkidneys (Rerolle et al., Kidney Int 58, 1841, 2000.). PAI-1 gene andprotein expression were assayed by quantitative RT-PCR and Western blot,respectively. Vesicular particles shed by cells into the culture media(e.g., exosomes) were collected by high-speed centrifugation (Wang etal., Nuc Acids Res 2010, 1-12 doi:10.1093/nar/gkq601, Jul. 7, 2010) andtotal RNA extracted from the pellet with TRIzol reagent (Invitrogen).RNA content of the vesicles was screened using PCR-based arrays of knownmicroRNA sequences (Qiagen).

Results: Conditioned media from bioactive kidney cell culturesattenuated the TGFβ1-induced increase in PAI-1 steady-state proteinlevels in mesangial cells, but did not affect steady state mRNA levels;an observation that is consistent with the mechanism by which microRNAsmodulate target genes. Based on the hypothesis that microRNAs can betransferred between cells through extracellular vesicle trafficking(Wang et al., supra 2010), we analyzed the conditioned media formicroRNA content and confirmed the presence of microRNA 30b-5p(miR-30b-5p), a putative inhibitor of PAI-1.

The data presented here suggest that bioactive kidney cells may modulatefibrosis directly through cell-to-cell transfer of miR-30b-5p to targetmesangial cells via exosomes. As a result of miR-30b-5p uptake bymesangial cells, TGFβ1-induced increases in steady-state PAI-1 proteinlevels are attenuated, a response that, in renal tissue, couldultimately reduce deposition of extracellular matrix within theglomerular space. Current work is underway to confirm that PAI-1 isindeed a direct target of miR-30b-5p.

FIGS. 14A-B show a western blot of PAI-1 and α-Actin (control) proteinexpression in human mesangial cells cultured for 24 hour in control(CTRL) or bioactive kidney cell conditioned media (CM) with (+) orwithout (−) TGFβ1 addition to the culture media. In CTRL cultures, TGFβ1increased PAI-1 protein expression. In CM cultures, the TGFβ1-inducedresponse was attenuated.

Secreted vesicles were analyzed for microRNAs that may be putativerepressors of PAI-1. Secreted vesicles from human and rat bioactivekidney cell CM were collected by high-speed centrifugation and assayedfor microRNA content using PCR-based arrays of known sequences.miR-449a, a putative regulator of PAI-1 (6), was identified. HRMC weretransiently transfected with miR-449a or not (CTRL). 24 hourspost-transfection cells were either exposed to 5 ng/ml TGFb1 (+) or not(−) for an additional 24 hours.

FIG. 16A shows a Western blot in which total protein was prepared andassayed for PAI-1 and bActin. miR-449a reduced steady-state PAI-1protein levels (compare lane 1 to lane 3) and induced levels of PAI-1protein were also lower in miR-449a transfected cultures (compare lane 2to lane 4). The data support the conclusion that excreted vesiclescontain miR-449a and uptake of miR-449a into mesangial cells reducesPAI-1 expression.

FIG. 16B depicts the microRNA, miR-30b-5p, which was also identified inthe PCR-based array and is a putative regulator of PAI-1 based onpredictive algorithms (http://mirbase.org—miRBase is hosted andmaintained in the Faculty of Life Sciences at the University ofManchester).

PAI-1 protein levels in glomeruli were examined in vivo after treatmentof CKD induced by 5/6 nephrectomy with bioactive renal cells.

FIGS. 17A-C show representative immunohistochemistry images of PAI-1(A-C) in Lewis rat kidneys that have undergone unilateral nephrectomy(A), 5/6 nephrectomy (B), or 5/6 nephrectomy with intra-renal deliveryof bioactive kidney cells (C). Accumulation of PAI-1 in the glomerulus(arrowheads) as a result of the 5/6 nephrectomy procedure (B) wasreduced as a result of treatment (C).

In a separate study, qRT-PCR was conducted on kidney tissue harvested atnecropsy and the relative gene expression values were plotted againstdays on study.

FIG. 17D shows that 5/6 nephrectomized rats (red squares) demonstratedmore robust expression of PAI-1 relative to those treated with bioactiverenal cells (blue diamonds) and sham-operated controls (greentriangles).

FIG. 17E shows representative Western blot analysis on kidney samplestaken at 3 and 6 months post-treatment. Treated tissues (Nx+Tx) of 5/6nephrectomized rats (Nx) had reduced the accumulation of PAI-1 andFibronectin (FN) protein (Kelley et al. 2010 supra).

The data support the conclusion that in vivo PAI-1 protein levels inglomeruli decrease after treatment of CKD induced by 5/6 nephrectomywith bioactive renal cells.

When taken together, Examples 12-13 support the hypothesis that onemechanism by which intra-renal delivery of bioactive kidney cellsimproves renal function might be via cell-cell transfer of componentsthat modulate fibrotic pathways in resident kidney cells.

Example 14—Secreted Factors from Bioactive Kidney Cells Attenuate NFKBSignaling Pathways

In this study, we investigated the role of NFκB pathways in theNKA-mediated attenuation of disease progression in the 5/6 nephrectomymodel and to identify properties of the bioactive kidney cells that maycontribute to regenerative outcomes through direct modulation of NFκBactivation. FIG. 17G depicts the canonical activation of the NFkBpathway by TNFα.

Materials and Methods: Remnant kidneys were harvested from Lewis rats inwhich a two-step 5/6 nephrectomy procedure was performed 6 weeks priorto being treated with B2+B4 in PBS (NKA prototype). NKA-treated (TX) oruntreated (UNTX) tissues were assayed for NFKB activation byimmunohistochemistry, RT-PCR, Western blot analysis, and electrophoresismobility shift assays (EMSA). Conditioned media (CM) collected from exvivo NKA cell cultures grown in serum- and supplement-free media wasused for in vitro functional assays. The human proximal tubule cell line(HK-2) was used as target cell type for molecular andimmunofluorsence-based assay readouts. Vesicular particles shed by cellsinto the culture media (exosomes) were collected by high-speedcentrifugation. Total RNA isolated from exosomes was screened usingPCR-based arrays of known microRNA sequences (Qiagen).

Results: Nuclear localization of the NFκB subunit, RelA/p65, wasobserved in remnant kidneys from 5/6 nephrectomized rats, suggestingactivation of inflammatory pathways in UNTX tissues. Preliminarycomparison with TX tissues by RT-PCR showed a decrease in RelA geneexpression, suggesting that NKA treatment may influence NFκB pathwayactivation through inhibition of RelA/p65 expression. This hypothesis issupported by the observation that CM attenuates TNFα-induced NFκBactivation in vitro, as evidenced by the reduced nuclear localization ofRelA/p65 in CM-exposed HK-2 cells (FIG. 17F) relative to that seen inresponse to Tumor Necrosis Factor-α (TNFα). Ongoing RT-PCR analyses ofNKA exosome microRNAs are investigating whether sequences known toinfluence NFκB pathways are present.

FIG. 17F shows a 2-hour exposure to NKA CM reduces nuclear localizationof NFκB p65 (green) in HK-2 compared to that observed in controlcultures pretreated with TNFα in immunofluorescent assays. In HK-2, NFkBp65 (green) localizes to the nucleus after a 30 minute exposure to TNFα(Control Media). However, pre-treatment of HK-2 cells with NKAConditioned Media for 2 hours prior to TNFα addition attenuated the NFkBp65 nuclear localization response. Nuclei are stained with DAPI (blue)and filamentous actin is stained with Alexa594-phalloidin (red) toassist in qualitatively assessing the robustness of NFκB nuclearlocalization (note the slightly diminished phalloidin borders inTNFα-treated control cells in the merged panels in the bottom row). Thecounterstaing provide reference for the NFkB localization in the mergedimages.

Immunohistochemistry for the NFkB p65 subunit in kidney tissues of Lewisrats reveals that animals with progressive CKD initiated by 5/6nephrectomy (panel B) have more robust nuclear localization of NFkB p65subunit, particularly in tubular epithelial cells (black arrowheads)relative to the non-progressive renal insufficiency initiated byunilateral nephrectomy in control animals (panel A). Tissues harvestedsix weeks post-nephrectomy. Magnification at 200×.

Panel C: Western blot analysis for NFkB p65 in the cytoplasmic (‘C’) andnuclear (‘N’) protein extracts of Lewis rat kidney tissue that haveundergone the 5/6 nephrectomy. Comparing weeks 1 and 13, where gtubulinlevels (loading control) are relatively consistent, nuclear NFkB p65increases over time, consistent with the immunohistochemistry results.

Panel D: Electrophoretic mobility shift assay (EMSA) on nuclear extractsconfirms that the NFkB that localizes to the nucleus following 5/6nephrectomy is activated for DNA binding. Lanes represent nuclearextracts prepared from two animals at each time point.

The NFkB pathway is progressively activated in the 5/6 nephrectomy modelof chronic kidney disease. Immunohistochemistry for the NFkB p65 subunitin kidney tissues of Lewis rats was performed.

FIGS. 18A-D reveal that animals with progressive CKD initiated by 5/6nephrectomy (panel B) have more robust nuclear localization of NFkB p65subunit, particularly in tubular epithelial cells (black arrowheads)relative to the non-progressive renal insufficiency initiated byunilateral nephrectomy in control animals (panel A). Tissues harvestedsix weeks post-nephrectomy. Magnification at 200×.

FIG. 18C shows Western blot analysis for NFkB p65 in the cytoplasmic(‘C’) and nuclear (‘N’) protein extracts of Lewis rat kidney tissue thathave undergone the 5/6 nephrectomy. Comparing weeks 1 and 13, wheregtubulin levels (loading control) are relatively consistent, nuclearNFkB p65 increases over time, consistent with the immunohistochemistryresults.

FIG. 18D shows an electrophoretic mobility shift assay (EMSA) on nuclearextracts and confirms that the NFkB that localizes to the nucleusfollowing 5/6 nephrectomy is activated for DNA binding. Lanes representnuclear extracts prepared from two animals at each time point. 1 mg ofnuclear protein was incubated with 5 ng of NFkB DNA binding site,electrophoresed on a 6% DNA retardation gel, then subsequently stainedwith ethidium bromide.

Intra-renal delivery of NKA cells reduces NFkB nuclear localization.Multiple defined subpopulations of renal cells have been isolated andassayed in vivo for bioactivity in improving renal function in the 5/6nephrectomy model of CKD (Presnell et al. 2010 supra). NKA cellsdemonstrated bioactivity whereas other subpopulations did not (Kelley etal. 2010 supra).

FIG. 18E shows that Lewis rats with established CKD that receivedintra-renal injection of NKA (A) or non-bioactive renal cells (B). Lewisrats with established CKD received intra-renal injection of NKA (A) ornon-bioactive renal cells (B). At 6 months post-treatment, tissues wereharvested and assayed by immunohistochemistry for the NFkB p65 subunit.Tissues from NKA-treated animals exhibited less nuclear localization ofNFkB p65, particularly in the proximal tubules, compared to tissues fromanimals treated with non-bioactive renal cells, suggesting that the NKAtreatment participated in attenuating the NFkB pathway activity in vivo.

Analysis of microRNA content of secreted vesicles isolated from humanand rat NKA conditioned media by high-speed centrifugation usingPCR-based arrays of known sequences identified several microRNA speciesthat may influence immune responses via NFkB based on literature reports(Marquez R T et al. (2010) Am J Physiol Gastrointest Liver Physiol298:G535; Taganov K D et al. (2006) Proc Natl Acad Sci USA 103:12481) orpredictive algorithms (http://mirbase.org—miRBase is hosted andmaintained in the Faculty of Life Sciences at the University ofManchester).

microRNA in vesicles Target mRNA miR-21 Pellino-1 (Marquez et al.)miR-146a IRAK1, TRAF6 (Taganov et al.) miR-124, miR-151 NFKB/RelA(miRBase)

The in vivo and in vitro findings provide insight on how bioactivekidney cells (NKA) might improve renal function in chronically-diseasedkidneys by modulating immune response pathways such as those affected byNFkB activation. Activated NFkB (p65 nuclear localization, particularlyin proximal tubule cells) is associated with the establishment ofchronic kidney disease in the 5/6 nephrectomy rodent model and wasattenuated by NKA treatment. The in vitro response of proximal tubulecells (HK-2) to NKA conditioned medium mimics the in vivo attenuation ofNFkB nuclear localization in response to NKA treatment. Putativemediators of cell-cell inhibition of NFkB activation (microRNAs) wereidentified in NKA conditioned medium. Taken together, these data supportthe hypothesis that one mechanism by which intra-renal delivery ofbioactive kidney cells improves renal function might be via cell-celltransfer of components, e.g., RNA, that modulate immune responses inresident kidney cells.

Example 15—Functional Evaluation of NKA Constructs

Renal cell populations seeded onto gelatin or HA-based hydrogels wereviable and maintained a tubular epithelial functional phenotype duringan in vitro maturation of 3 days as measured by transcriptomic,proteomic, secretomic and confocal immunofluorescence assays. Toinvestigate a potential mechanism by which NKA Constructs could impact adisease state, the effect of conditioned media on TGF-β signalingpathways related to tubulo-interstitial fibrosis associated with CKDprogression was evaluated. Conditioned medium was observed to attenuateTGF-β-induced epithelial-mesenchymal transition (EMT) in vitro in ahuman proximal tubular cell line (HK2).

Materials and Methods.

Biomaterials. Biomaterials were prepared as beads (homogenous, sphericalconfiguration) or as particles (heterogenous population with jaggededges). Gelatin beads (Cultispher S and Cultispher GL) manufactured byPercell Biolytica (Åstorp, Sweden) were purchased from Sigma-Aldrich(St. Louis, Mo.) and Fisher Scientific (Pittsburgh, Pa.), respectively.Crosslinked HA and HA/gelatin (HyStem™ and Extracel™ from GlycosanBioSystems, Salt Lake City, Utah) particles were formed from lyophilizedsponges made according to the manufacturer's instructions. Gelatin(Sigma) particles were formed from crosslinked, lyophilized sponges.

PCL was purchased from Sigma-Aldrich (St. Louis, Mo.). PLGA 50:50 waspurchased from Durect Corp. (Pelham, Al.). PCL and PLGA beads wereprepared using a modified double emulsion (W/O/W) solvent extractionmethod. PLGA particles were prepared using a solvent casting porogenleaching technique. All beads and particles were between 65 and 355microns when measured in a dry state.

Cell isolation, preparation and culture. Cadaveric human kidneys wereprocured through National Disease Research Institute (NDRI) incompliance with all NIH guidelines governing the use of human tissuesfor research purposes. Canine kidneys were procured from a contractresearch organization (Integra). Rat kidneys (21 day old Lewis) wereobtained from Charles River Labs (MI). The preparation of primary renalcell populations (UNFX) and defined sub-populations (B2) from whole rat,canine and human kidney has been previously described (Aboushwareb etal. World J Urol 26(4):295-300; 2008; Kelley et al. supra 2010; Presnellet al. WO/2010/056328). In brief, kidney tissue was dissociatedenzymatically in a buffer containing 4.0 units/mL dispase (Stem CellTechnologies, Inc., Vancouver BC, Canada) and 300 units/ml collagenaseIV (Worthington Biochemical, Lakewood N.J.), then red blood cells anddebris were removed by centrifugation through 15% iodixanol (Optiprep®,Axis Shield, Norton, Mass.) to yield UNFX. UNFX cells were seeded ontotissue culture treated polystyrene plates (NUNC, Rochester N.Y.) andcultured in 50:50 media, a 1:1 mixture of high glucose DMEM:KeratinocyteSerum Free Medium (KSFM) containing 5% FBS, 2.5 μg EGF, 25 mg BPE, 1XITS (insulin/transferrin/sodium selenite medium supplement), andantibiotic/antimycotic (all from Invitrogen, Carlsbad Calif.). B2 cellswere isolated from UNFX cultures by centrifugation through a four-stepiodixanol (OptiPrep; 60% w/v in unsupplemented KSFM) density gradientlayered specifically for rodent (16%, 13%, 11%, and 7%), canine (16%,11%, 10%, and 7%), or human (16%, 11%, 9%, and 7%) (Presnell et al.WO/2010/056328; Kelley et al. supra 2010). Gradients were centrifuged at800×g for 20 minutes at room temperature (without brake). Bands ofinterest were removed via pipette and washed twice in sterile phosphatebuffered saline (PBS).

Cell/biomaterial composites (NKA Constructs). For in vitro analysis ofcell functionality on biomaterials, a uniform layer of biomaterials(prepared as described above) was layered onto one well of a 6-well lowattachment plate (Costar #3471, Corning). Human UNFX or B2 cells(2.5×10⁵ per well) were seeded directly onto the biomaterial. Forstudies of adherence of canine cells to biomaterials, 2.5×10⁶ UNFX cellswere seeded with 50 μl packed volume of biomaterials in a non-adherent24-well plate (Costar #3473, Corning). After 4 hours on a rockingplatform, canine NKA Constructs were matured overnight at 37° C. in a 5%CO₂ incubator. The next day, live/dead staining was performed using alive/dead staining assay kit (Invitrogen) according to themanufacturer's instructions. Rat NKA Constructs were prepared in a 60 ccsyringe on a roller bottle apparatus with a rotational speed of 1 RPM.

For the transcriptomic, secretomic, and proteomic analyses describedbelow, NKA Constructs were matured for 3 days. Cells were then harvestedfor transcriptomic or proteomic analyses and conditioned media wascollected for secretomic profiling.

Functional analysis of tubular cell associated enzyme activity. CanineNKA Constructs (10 μl loose packed volume) in 24-well plates wereevaluated using an assay for leucine aminopeptidase (LAP) activityadapted from a previously published method (Tate et al. Methods Enzymol113:400-419; 1985). Briefly, 0.5 ml of 0.3 mM L-leucine p-nitroanalide(Sigma) in PBS was added to NKA Constructs for 1 hour at roomtemperature. Wells were sampled in duplicate and absorbance at 405 nmrecorded as a measure of LAP activity. LLC-PK1 cell lysate (AmericanType Culture Collection, or ATCC) served as the positive control.

Transcriptomic profiling. Poly-adenylated RNA was extracted using theRNeasy Plus Mini Kit (Qiagen, Calif.). Concentration and integrity wasdetermined by UV spectrophotometry. cDNA was generated from 1.4 μgisolated RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen).Expression levels of target transcripts were examined by quantitativereal-time polymerase chain reaction (qRT-PCR) using commerciallyavailable primers and probes (Table 15.1) and an ABI-Prism 7300 RealTime PCR System (Applied Biosystems, CA). Amplification was performedusing TaqMan Gene Expression Master Mix (ABI, Cat #4369016) and TATA BoxBinding Protein gene (TBP) served as the endogenous control. Eachreaction consisted of 10 μl Master Mix (2×), 1 μl Primer and Probe (20×)and 9 μl cDNA. Samples were run in triplicate.

TABLE 15.1 Human TaqMan Primers/Probes Gene Abbrv. Marker TaqMan Cat #Aquaporin 2 AQP2 Distal Collecting Hs00166640_m1 Duct Tubule EpithelialCadherin/Cadherin 1. Type 1 CDH1/ECAD Distal Tubule Hs00170423_m1Neuronal Cadherin/Cadherin 2. Type 2 CDH2/NCAD Proximal TubuleHs00169953_m1 Cubilin, Intrinsic Factor-Cobalamin Receptor CUBN ProximalTubule Hs00153607_m1 Nephrin NPHS1 Glomerular/Podocyte Hs00190466_m1Podocin NPHS2 Glomerular/Podocyte Hs00922492_m1 Erythropoietin EPOKidney Interstitum Hs01071097_m1 Cytochrome P450. Family 24, SubfamilyA, CYP2R1 Proximal Tubule Hs01379776_m1 Polypeptide 1/Vitamin D24-Hydroxylase Vascular Endothelial Growth Factor A VEGFAEndothelial/Vascular Hs00900055_m1 Platelet/Endothelial Cell AdhesionMolecule PECAM1 Endothelial/Vascular Hs00169777_m1 Smooth Muscle MyosinHeavy Chain MYHC/ Smooth Muscle Hs00224610_m1 SMMHC Calponin CNN1 SmoothMuscle Hs00154543_m1 TATA Box Binding Protein TBP Endogenous ControlHs99999910_m1

Secretomic profiling. Conditioned medium from human NKA Constructs wascollected and frozen at −80° C. Samples were evaluated for biomarkerconcentration quantitation. The results for a given biomarkerconcentration in conditioned media were normalized relative to theconcentration of the same biomarker in conditioned media from controlcultures (2D culture without biomaterial) and expressed as a unitlessratio.

Proteomic profiling. Protein from three independent replicates wasextracted from cell/biomaterial composites and pooled for analysis by 2Dgel electrophoresis. All reagents were from Invitrogen. Isoelectricfocusing (IEF) was conducted by adding 30 μg of protein resuspended in200 μl of ZOOM 2D protein solubilizer #1 (Cat # ZS10001), ZOOM carrierampholytes pH 4-7 (Cat # ZM0022), and 2M DTT (Cat #15508-013) to pH 4-7ZOOM IEF Strips (Cat # ZMO012). Following electrophoresis for 18 hoursat 500V, IEF strips were loaded onto NuPAGE Novex 4-12% Bis-Tris ZOOMIPG well gels (Cat # NP0330BOX) for SDS-PAGE separation andelectrophoresed for 45 min at 200V in IVIES buffer (Cat # NP0002).Proteins were visualized using SYPRO Ruby protein gel stain (Cat #S-12000) according to the manufacturer's instructions.

Confocal microscopy. NKA Constructs prepared from human or rat UNFX orB2 cells were matured for 3 days and then fixed in 2% paraformaldehydefor 30 minutes. Fixed NKA Constructs were blocked and permeabilized byincubation in 10% goat serum (Invitrogen) in D-PBS (Invitrogen)+0.2%Triton X-100 (Sigma) for 1 hour at room temperature (RT). Forimmunofluorescence, NKA Constructs were labeled with primary antibodies(Table 15.2) at a final concentration of 5 μg/ml overnight at RT.Labeled NKA constructs were washed twice with 2% goat serum/D-PBS+0/2%Triton X-100 and incubated with goat or rabbit TRITC conjugatedanti-mouse IgG2A (Invitrogen) secondary antibody at 5 μg/ml. For doublelabeling with DBA (Dolichos biflorus agglutinin), NKA constructcandidates were further incubated with FITC conjugated DBA (Vector Labs)diluted to 2 mg/ml in 2% goat serum/D-PBS+0.2% Triton X-100 for 2 hrs atRT.

TABLE 15.2 Antibody Source Manufacturer Catalog # Target IgG1 ctrl MouseBD 557273 Background control IgG ctrl goat Invitrogen 026202 Backgroundcontrol IgG ctrl rabbit Invitrogen 026102 Background control N-CadherinMouse BD 610920 Proximal tubules E-Cadherin Mouse BD 610182 Distaltubules Cubilin goat Santa Cruz Sc-20609 Proximal tubules (A-20) GGT-1Rabbit Santa Cruz Sc-20638 Tubular epithelial Megalin Rabbit Santa CruzSc-25470 Proximal tubules

Samples were washed twice with D-PBS and optically sectioned using aZeiss LSM510 laser scanning confocal system (Cellular Imaging Core, WakeForest Baptist Medical Center) running LSM Image software (Zeiss) orwith a Pathway 855 confocal microscope (BD Biosciences).

Analysis of TGF-β mediated EMT in HK2 cells. HK2 cells (ATCC) werecultured in 50:50 media in fibronectin or collagen (IV) coated culturedishes (BD Biosciences). For EMT assays, HK2 cells were seeded in24-well collagen (IV) coated plates at 70-80% confluency with 50:50media or conditioned media collected from either two dimensional (2D)human UNFX cultures or NKA Constructs made with human UNFX that werematured for 3 days prior to media collection. TGF-β induction wasinitiated by adding 10 ng/ml to the culture media 3 days prior toisolating RNA from the cells for the EMT assay. EMT was monitored byqRT-PCR by analyzing the relative expression of E-cadherin (anepithelial marker) and calponin (mesenchymal marker) at the end of thethree day incubation period. RNA was prepared from harvested HK2 cellsfor TaqMan qRT-PCR analysis as described above. Statistical analysis wasdone using standard two tailed Student's t-test assuming equal variancefor each sample. Confidence intervals of 95% (p-value<0.05) and 99%(p-value<0.01) were used to determine statistical significance.

In vivo implantation of acellular biomaterials and NKA Constructs. Lewisrats (6 to 8 weeks old) were purchased from Charles River (Kalamazoo,Mich.). All experimental procedures were performed under PHS and IACUCguidelines of the Carolinas Medical Center. Under isoflurane anesthesia,female Lewis rats (approximately 2 to 3 months old) underwent a midlineincision, and the left kidney was exposed. 35 μl of packed biomaterials(acellular biomaterial or NKA Construct) were introduced bymicroinjection into the renal parenchyma. Two injection trajectorieswere used: (i) from each pole toward the cortex (referred to as corticalinjection), or (ii) from the renal midline toward the pelvis (referredto as medullary injection). Rats were sacrificed at 1, 4, or 8 weekspost-injection. No early deaths occurred. Study design for the acellularimplantation study is presented in Table 15.3 (ND=not done).

Renal Histology. Representative kidney samples were collected and placedin 10% buffer formalin for 24 hours. Sections were dehydrated inascending grades of ethanol and embedded in paraffin. Sections (5 μm)were cut, mounted on charged slides, and processed for hematoxylin andeosin (H&E), Masson's trichrome and Periodic Acid Schiff (PAS) stainingin accordance with standard staining protocols (Prophet et al., ArmedForces Institute of Pathology: Laboratory methods in histotechnology.Washington, DC: American Registry of Pathology; 1992). Digitalmicrophotographs were captured at total magnification of ×40, ×100 and×400 using a Nikon Eclipse 50i microscope fitted with a Digital Sight(DS-U1) camera. Renal morphology changes were assessed by commonly used(Shackelford et al. Toxicol Pathol 30(1):93-96; 2002) severity gradeschemes (grades 1, 2, 3, 4), to which descriptive terms (minimal, mild,moderate, marked/severe) were applied to describe the degree ofglomerulosclerosis, tubular atrophy and dilatation, tubular casts, andinterstitial fibrosis, and inflammation observed.

TABLE 15.3 Study design for evaluating acellular biomaterials in healthyadult Lewis rat kidneys Time in vivo Biomaterial: 1 week 4 weeks PCLBeads n = 1 n = 1 Gelatin Beads n = 1 ND Gelatin Particles n = 1 n = 1HA/Gelatin Particles n = 2 ND HA Particles n = 1 n = 1 PLGA Particles n= 1 ND PLGA Beads n = 1 ND

Results

Response of mammalian kidney tissue to injection of biomaterials intothe renal parenchyma. Biomaterials were analyzed for potential use inrenal cell/biomaterial composites by direct injection into healthy ratkidneys (Table 15.3). Tissue responses were evaluated by measuring thedegree of histopathology parameters (inflammation, fibrosis, necrosis,calcification/mineralization) and biocompatibility parameters(biomaterial degradation, neo-vascularization, and neo-tissue formation)at 1 and 4 weeks post-injection.

FIGS. 19A-B show in vivo evaluation of biomaterials at 1 weekpost-implantation. Trichrome X10 low power image of kidney cross sectionshowing biomaterial aggregate. Trichrome X40: Close-up of biomaterialaggregate. H&E X400: High magnification image of biomaterial aggregateto evaluate extent of cell/tissue infiltration. Each kidney was injectedat two locations as described in Materials and Methods. At 1 weekpost-implantation, the host tissue responses elicited by eachbiomaterial tested were generally similar; however, gelatin hydrogelsappeared to elicit less intense histopathological and more biocompatibleresponses.

FIG. 19C shows in vivo evaluation of biomaterials at 4 weekspost-implantation. At 4 weeks post-implantation, the severity ofhistopathology parameters in tissues injected with HA or gelatinparticles were qualitatively reduced compared to 1 weekpost-implantation. Gelatin particles were nearly completely resorbed andless giant cell reaction was observed than in tissues that received HAparticles. In most cases where biomaterials were injected via themedullary injection trajectory (e.g., deeper into the medulla/pelvis),undesirable outcomes including obstruction leading to hydronephrosis,inflammatory reactions of greater severity, and renal arteriolar andcapillary micro-embolization leading to infarction was observed (datanot shown).

Assessing functional phenotype of therapeutically-relevant renal cellpopulations with biomaterials. Therapeutically-relevant renal cellpopulations (UNFX) that extended survival and increased renal functionin a rodent model of chronic kidney disease after direct injection intorenal parenchyma have been characterized (Presnell et al.WO/2010/056328; Kelley et al. supra 2010) and methods for theirisolation, characterization, and expansion have been developed andtranslated across multiple species (Presnell et al. 2010 supra). Toassess whether UNFX cells adhere to, remain viable, and retain apredominantly tubular, epithelial phenotype when incorporated into NKAConstructs, transcriptomic, secretomic, proteomic, and confocalimmunofluorescence microscopy analyses were conducted on NKA Constructsproduced from UNFX cells and various biomaterials.

Adherence and viability. Canine-derived UNFX cells were seeded withgelatin beads, PCL beads, PLGA beads, HA particles, and HA/gelatinparticles as described (3 NKA Constructs per biomaterial). Celldistribution and viability were assessed one day after seeding bylive/dead staining

FIGS. 20A-D show live/dead staining of NKA constructs seeded with canineUNFX cells (A=gelatin beads; B=PCL beads; C=HA/gelatin particles; D=HAparticles). Green indicates live cells; red indicates dead cells. (A)Gelatin beads; (B) PCL beads; (C) HA/gelatin particles; and (D) HAparticles. Viable cells may be observed on all hydrogel-based NKAConstructs.

UNFX cells adhered robustly to naturally-derived, hydrogel-basedbiomaterials such as gelatin beads and HA/gelatin particles (blackarrows in A, D), but showed minimal adherence to synthetic PCL (B) orPLGA beads (not shown). Cells did not adhere to HA particles (C) butshowed evidence of bioresponse (i.e., spheroid formation). Functionalviability of the seeded UNFX cells on hydrogel-based NKA Constructs wasconfirmed by assaying for leucine aminopeptidase, a proximaltubule-associated hydrolase (data not shown).

Transcriptomic profiling. The gene expression profiles of human UNFXcells in hydrogel-based NKA Constructs (3 NKA Constructs perbiomaterial) and parallel 2D cultures of UNFX cells were compared byquantitative transcriptomic analysis.

FIGS. 20E-G show transcriptomic profiling of NKA constructs. TC: primaryhuman UNFX cells cultured in 2D. Gelatin: NKA Construct composed ofhuman UNFX cells and gelatin hydrogel. HA-Gel: NKA Construct composed ofhuman UNFX cells and HA/gelatin particles. qRT-PCR data presented ingraphical and tabular format. Transcripts examined fell into fourprincipal categories: (i) Tubular: aquaporin 2(AQ2), E-cadherin (ECAD),erythropoietin (EPO), N-cadherin (NCAD), Cytochrome P450, Family 24,Subfamily A, Polypeptide 1—aka Vitamin D 24-Hydroxylase (CYP), cubilin,nephrin; (ii) Mesenchymal: calponin (CNN1), smooth muscle myosin heavychain (SMMHC); (iii) Endothelial: vascular endothelial growth factor(VEGF), platelet endothelial cell adhesion molecule (PECAM); and (iv)Glomerular: podocin. Overall, tubular marker expression was comparablebetween hydrogel-based NKA Constructs and 2D UNFX cultures. Similarly,endothelial markers (VEGF and PECAM) were comparable. In contrast, theglomerular marker podocin exhibited significant variation among NKAConstructs. Podocin levels in HA/gelatin-based NKA Constructs were mostcomparable with those observed in 2D UNFX cultures. Interestingly,mesenchymal marker (CNN1 and SMMHC) expression was significantlydown-regulated (p<0.05) in hydrogel-based NKA Constructs relative to 2DUNFX cultures, suggesting that fibroblastic sub-populations of UNFX maynot propagate as well in the hydrogel-based NKA Constructs in the renalmedia formulation.

Secretomic profiling. NKA Constructs were produced with human UNFX andB2 cells and gelatin or HA/gelatin hydrogel (one NKA Construct perbiomaterial per cell type=4 NKA Constructs total).

FIGS. 21A-B show the secretomic profiling of NKA Constructs. Data ispresented as a 3D:2D ratio. NKA Constructs were produced from human UNFXor B2 cells and gelatin (Hydrogel 1) or HA/gelatin (Hydrogel 2)hydrogels as described in Materials and Methods. Secretomic profilingwas performed on conditioned media from NKA Constructs matured for 3days and compared with parallel 2D cultures of human UNFX or B2 cells bycalculating the ratio of analyte expression of NKA Constructs(three-dimensional, or 3D, culture) to 2D culture (3D:2D ratio). Foreach of the three NKA Constructs seeded with UNFX cells, the 3D:2Dratios were at or close to 1, suggesting that the seeding process and 3days of maturation on these biomaterials had little impact on thesecretomic profile of UNFX cells. For NKA Constructs seeded with B2cells, a similar result of a 3D:2D ratio at or near 1 was observed,providing additional evidence that the seeding process and 3 days ofmaturation on these biomaterials had little impact on the secretomicprofile of therapeutically-relevant renal cells.

Proteomic profiling. Proteomic profiles of a given cell or tissue areproduced by separating total cellular proteins using 2D gelelectrophoresis and have been used to identify specific biomarkersassociated with renal disease (Vidal et al. Clin Sci (Lond)109(5):421-430; 2005).

FIGS. 22A-B show proteomic profiling of NKA Constructs. NKA Constructswere produced with human UNFX cells and biomaterials as indicated.Proteins in total protein extracts were separated by 2D gelelectrophoresis as described in Materials and Methods. In thisexperiment, proteomic profiling was used to compare protein expressionin human UNFX cells in NKA Constructs (gelatin or HA/gelatinhydrogel-based, 3 NKA Constructs per biomaterial) and in 2D tissueculture. The proteome profiles of total protein isolated from NKAConstructs or 2D cultures of UNFX cells were essentially identical,providing additional evidence that the seeding process and 3 daysmaturation on these biomaterials had little impact on the proteomesexpressed by UNFX cells.

Confocal microscopy. Retention of the tubular epithelial phenotype ofrat and human B2 cells (Presnell et al. 2010 supra) in NKA Constructswas evaluated by confocal imaging of established biomarkers: FIGS. 23A-Cshow confocal microscopy of NKA Constructs. Confocal microscopy of NKAConstructs produced with human (A) or rat (B, C) B2 cells and gelatinhydrogel. (A) E-cadherin (red—solid white arrows), DBA (green—dashedgreen arrows) and gelatin hydrogel bead is visible with DIC optics. (B)DNA visualized with DAPI staining (blue—solid white arrows) and each ofthe following markers in green (dashed white arrows): IgG control,N-cadherin, E-cadherin, cytokeratin 8/18/18, DBA. (C) double-labelingimages of markers and colors as indicated. E-cadherin and DBA in humanNKA Constructs and E-cadherin, DBA, N-cadherin, cytokeratin 8/18/19,gamma glutamyl transpeptidase (GGT-1), and megalin in rat NKAConstructs. Optical sectioning of confocal images also allowedevaluation of the extent of cell infiltration into the biomaterial afterseeding and 3 days of maturation. B2 cells in human and rat NKAConstructs exhibited expression of multiple tubular epithelial markers.Optical sectioning revealed minimal cell infiltration of the hydrogelconstruct, with cells generally confined to the surface of thebiomaterial.

In vivo responses to implantation of NKA construct prototypes. Based onthe in vivo responses to biomaterial injection into renal parenchyma andthe in vitro phenotype and functional characterization of UNFX and B2cells in NKA Constructs described above, gelatin hydrogel was selectedto evaluate the in vivo response to NKA Construct injection into renalparenchyma in healthy Lewis rats. NKA Constructs were produced fromsyngeneic B2 cells and implanted into two animals, which were sacrificedat 1, 4, and 8 weeks post-implantation. All animals survived toscheduled necropsy when sections of renal tissues were harvested,sectioned, and stained with Trichrome, hematoxylin and eosin (H&E), andPeriodic Acid Schiff (PAS).

FIGS. 24A-B show in vivo evaluation of NKA Constructs at 1 and 4 weekspost-implantation. Trichrome X10 low power image of kidney cross sectionshowing biomaterial aggregate. Trichrome X40: Close-up of biomaterialaggregate. H&E/PAS X400: High magnification image of biomaterialaggregate to evaluate extent of cell/tissue infiltration. Each kidneywas injected at two locations as described in Materials and Methods.

FIG. 24A shows in vivo evaluation of NKA Constructs at 1 weekpost-implantation. At 1 week post injection, gelatin beads were presentas focal aggregates (left panel, circled area) of spherical and porousmaterial staining basophilic and surrounded by marked fibro-vasculartissue and phagocytic multi-nucleated macrophages and giant cells.Fibrovascular tissue was integrated within the beads and displayedtubular epithelial components indicative of neo-kidney tissue formation.Additionally, tubular and vasculoglomerular structures were identifiedby morphology (PAS panels).

FIG. 24B shows in vivo evaluation of NKA Constructs at 4 weekspost-implantation. By 4 weeks post-injection, the hydrogel wascompletely resorbed and the space replaced by progressive renalregeneration and repair with minimal fibrosis (note the numerousfunctional tubules within circled area of 4-week Trichrome panel).

FIGS. 25A-D show in vivo evaluation of NKA Construct at 8 weekspost-implantation. Trichrome X10 low power image of kidney cross sectionshowing biomaterial aggregate. Trichrome X40: Close-up of biomaterialaggregate. H&E/PAS X400: High magnification image of biomaterialaggregate to evaluate extent of cell/tissue infiltration. (A) Moderatechronic inflammation (macrophages, plasma cells and lymphocytes),moderate numbers of hemosiderin-laden macrophages (chronic hemorrhagedue to injection) with marked fibrovascular response (blue stained byMasson's trichrome—black arrows).; (B) Higher magnification (trichromestained, ×400) of boxed area of (A) showing regenerative responseinduction consistent with neo-kidney tissue formation (C) Representativeof adjacent (normal) kidney parenchyma showing typical corticalglomeruli morphology HE, ×400); (D) HE stained section, ×400 comparingnew glomeruli morphology observed in treatment area vs. FIG. 25C.

FIGS. 25A-D show in vivo evaluation of NKA Construct at 8 weekspost-implantation. At 8 weeks post-implantation, evidence of neo-kidneylike tissue formation was observed, consistent with induction of earlyevents in nephrogenesis. Comparison of the area of regenerativeinduction (B, D) with adjacent cortical parenchyma (C) showed presenceof multiple S-shaped bodies and newly formed glomeruli.

Effect of conditioned media from NKA Constructs on TGF-β induced EMT inHK2 cells. The development of tubulo-interstitial fibrosis during theprogression of CKD is associated with TGF-β mediated EMT of tubularepithelial cells (Zeisberg et al. Am J Pathol 160(6):2001-2008; 2002).Also, attenuation of TGF-β pathways was observed in vivo in a rodentmodel of progressive CKD where survival was extended and renal functionimproved by treatment with UNFX and B2 cells (Presnell et al.WO/2010/056328). The human proximal tubular cell line HK2 has been wellestablished as an in vitro model system to test the stimulatory orinhibitory effects of small molecules or proteins on TGF-β induced EMT(Dudas et al. Nephrol Dial Transplant 24(5):1406-1416; 2009; Hills etal. Am J Physiol Renal Physiol 296(3):F614-621; 2009). To investigate apotential mechanism by which NKA Constructs might affect renal tissueresponses post-implantation, conditioned medium collected from NKAConstructs produced with UNFX cells and hydrogel was evaluated in theHK2 EMT assay system.

FIG. 26 shows conditioned medium from NKA Constructs attenuates TGF-βinduced EMT in HK2 cells in vitro. EMT is monitored by quantitating therelative expression of ECAD (epithelial) and CNN1 (mesenchymal) markers.HK2 cells were cultured in 50:50 media (Control and TGFB Controlsamples) or conditioned medium (CM) from 2D cultures of human UNFX cells(TC) or NKA Constructs produced from human UNFX cells and either Gelatinor HA/Gelatin as indicated. To induce EMT, 10 ng/ml TGF-β was added toeach sample (except Control) for 3 days prior to assay. When HK2 cellswere cultured in 50:50 media (Control), ECAD (epithelial marker) wasexpressed at higher levels than CNN1 (mesenchymal marker). When TGF-β isadded to the media for 3 days (TGFB Control), ECAD expression wassignificantly down-regulated with a concomitant up-regulation of CNN1,consistent with induction of an EMT event. Conditioned medium from 2DUNFX cell cultures significantly (p<0.05 for both ECAD and CNN1)attenuated the EMT response of HK2 cells to TGF-β (TC CM). Conditionedmedium from NKA Constructs (Gelatin CM and HA/Gelatin CM) alsoattenuated the EMT response to TGF-β; however the overall effect wasless than that observed with conditioned medium from 2D UNFX cellcultures (significant—p<0.05—for ECAD with both NKA Constructs andtrending toward control though not statistically significant for CNN1).Additional mesenchymal markers were screened and yielded similar results(data not shown). These data suggest that NKA Constructs couldpotentially affect TGF-β pathways associated with tubulo-interstitialfibrosis in vivo in a manner similar to that observed with cell-basedtreatment (Presnell et al. WO/2010/056328). These data also suggest thatthe in vitro EMT assay has potential application forscreening/optimizing/monitoring the biotherapeutic efficacy of NKAConstructs if in vivo responses can be demonstrated to have astatistically significant association with in vitro EMT responses,thereby potentially reducing the need for time consuming and expensivein vivo assays.

This study investigated the responses of mammalian renal parenchyma toimplantation of synthetic and natural biomaterials, both acellular andas bioactive renal cell/biomaterial composites (i.e., NKA Constructs). Acombination of in vitro functional assays and in vivo regenerativeoutcomes were analyzed to functionally screen candidate biomaterials forpotential incorporation into a NKA construct prototype. Implantation ofacellular hydrogel-based biomaterials into renal parenchyma (FIG. 19)was typically associated with minimal fibrosis or chronic inflammationand no evidence of necrosis by 4 weeks post-implantation. Moderatecellular/ tissue in-growth and neo-vascularization was observed, withminimal remnant biomaterial. Based on these in vivo data, hydrogel-basedbiomaterials were selected to produce NKA Constructs with which toevaluate in vitro biofunctionality and in vivo regenerative potential.In vitro confirmation of material biocompatibility was provided throughlive/dead analysis of NKA Constructs (FIG. 20). Gelatin-containinghydrogels were associated with robust adherence of primary renal cellpopulations. Phenotypic and functional analysis of NKA Constructsproduced from bioactive primary renal cell populations (UNFX or B2) andhydrogel biomaterials was consistent with continued maintenance of atubular epithelial cell phenotype. Transcriptomic, secretomic,proteomic, and confocal microscopy analyses of NKA Construct confirmedno significant differences relative to primary renal cells seeded in 2Dculture. Finally, implantation of hydrogel-based NKA construct into therenal parenchyma of healthy adult rodents was associated with minimalinflammatory and fibrotic response and regeneration of neo-kidney liketissue by 8 weeks post-implantation.

Taken together, these data provide evidence suggesting that aregenerative response was induced in vivo by NKA Constructs. Thesestudies represent the first in vivo, intra-renal investigations of thebiological response of mammalian kidney to implantation of atherapeutically-relevant primary renal cell/biomaterial composite.Observed results are suggestive that NKA Constructs have the potentialto both facilitate regeneration of neo-kidney tissue and attenuatenon-regenerative (e.g., reparative healing) responses.

Bioresponse of Mammalian Kidney to Implantation of Polymeric Materials.In another study, host tissue responses to intra-renal injection ofnatural and synthetic biomaterials in rodent kidney were investigated toevaluate candidate biomaterials for forming cell/biomaterial compositeswith bioactive renal cell populations (Presnell et al. supra 2010).Methods: Natural biomaterials included gelatin and hyaluronic acid (HA).Synthetic biomaterials included polycaprolactone (PCL) andpoly-lactic-co-glycolic acid (PLGA). Candidate biomaterials wereevaluated in two discrete physical conformations: homogenous, sphericalbeads or heterogenous and non-uniform particles. PCL and PLGA beads wereprepared using a modified double emulsion (water/oil/water) solventextraction method. Gelatin beads were purchased (Cultispher-S®,Sigma-Aldrich, St. Louis, Mo.). PLGA particles were prepared using asolvent casting porogen leaching technique; gelatin and HA particleswere prepared from cross-linked, lyophilized foam. Two injections of 35μl of loosely packed biomaterials were delivered to the left kidneyparenchyma of 3 month old Lewis rats. Histopathologic evaluation offormalin-fixed sections of kidney tissue at 1 and 4 weeks post-injectionwas conducted using a semi-quantitative grading severity scale from 0(absent) to 4 (marked) of inflammation, tissue/cellular in-growth,neo-vascularization, material degradation, and fibro-cellular responses.Overall scores were calculated as the ratio of % positive to % negativeresponse (the higher the overall score the superior outcome).

Results. Histopathologic evaluation performed on biomaterialcandidates—representative 40× images of kidneys harvested 1 weekpost-implantation, sections stained with Masson's Trichrome (data notshown). Materials composed of polymers of natural origin, such asgelatin and HA were associated with milder fibro-cellular response andchronic inflammation, and greater cellular in-growth,neo-vascularization, biomaterial degradation, and necessary inflammationrequired for tissue healing and integration when compared to thesynthetic biomaterials, such as PLGA and PCL (organized fibrousencapsulation). Summary of histopathologic evaluation scoring. Scoreswere averaged by material composition (mean±SD). The synthetic materials(PLGA and PCL) scored the lowest, and gelatin materials generally scoredhigher than HA materials. This trend is most pronounced at the 4 weektime point. Due to factors unrelated to the material injections, not allthe samples tested at 1 week were available for analysis at 4 weeks. Thenumber of samples that are included in the gelatin, HA, and syntheticgroups are 3, 4, 3 at 1 week and 2, 3, 1 at 4 weeks, respectively.

Biomaterials of natural origin (e.g., gelatin or HA) delivered byinjection to healthy renal parenchyma elicited tissue responses wereless pathologic at 4 weeks post-injection than those of synthetic originas measured by semi-quantitative histopathologic evaluation.

Example 16—Hypoxic Exposure of Cultured Human Renal Cells InducesMediators of Cell Migration and Attachment and Facilitates the Repair ofTubular Cell Monolayers in Vitro

The role of oxygen tension in the isolation and function of a selectedpopulation of renal epithelial cells (B2) with demonstrated therapeuticfunction in models of chronic kidney disease (CKD) was investigated.This study examined whether low oxygen exposure during processing alterscomposition and function of selected human selected renal cells (SRCs)or bioactive renal cells (BRCs). Upon exposure to 2% Oxygen, thefollowing was observed: an alteration of the distribution of cellsacross a density gradient (see Presnell et al. WO 10/056328 incorporatedherein by reference in its entirety), improvement in overallpost-gradient yield, modulation of oxygen-regulated gene expression(previously reported in Kelley et al. supra (2010)), increasedexpression of erythropoietin, VEGF, HIF1-alpha, and KDR(VEGFR2).In-process exposure to low oxygen enhances the ability of selectedbioactive renal cells to repair/regenerate damaged renal tubules.

FIG. 27 depicts the procedure for exposing cells to low oxygen duringprocessing. FIG. 28 shows that upon exposure to 2% Oxygen, the followingwas observed: alters distribution of cells across a density gradient,improves overall post-gradient yield. Hypoxic exposure (<3%) increasedrecovery of cultured human CKD-derived renal cells from iodixanol-baseddensity gradients relative to atmospheric oxygen tension (21%) (96% vs.74%) and increased the relative distribution of selected cells (B2) intohigh-density (>9% iodixanol) fractions (21.6% vs. 11.2%).

Competitive in vitro assays demonstrated that B2 cells pre-exposed for24 hours to hypoxic conditions were more proficient in repairing damagedrenal proximal tubular monolayer cultures than B2 cells cultured at 21%oxygen tension, with 58.6%±3% of the repair occurring within two hoursof injury.

FIG. 29A depicts an assay developed to observe repair of tubularmonolayers in vitro. 1. Cells are labeled with fluorescent dyes (2%oxygen, 21% oxygen, and HK2 tubular cells). 2. The tubular cellmonolayer was established and wounded. 3. Oxygen-exposed labeled cellsare added (2% and 21% exposed cells). They are seeded equally at20,000/cm2. Culturing is in serum-free media at 5% O2 for 24 hrs. 4.Cells that repair wounding are quantified. FIG. 29B—Quantitative ImageAnalysis (BD Pathway 855 Biolmager)—red circles=cells cultured 2% O2,blue circles=21% O2. FIG. 29C—it was observed that 2% oxygen-inducedcells attached more rapidly (2 hrs) and sustained a mild advantage for24 hrs. Cells induced with 2% oxygen were more proficient at repair oftubular epithelial monolayers.

FIG. 30A depicts an assay developed to observe repair of tubularmonolayers in vitro. 1. Cells were labeled with fluorescent dyes. 2. Thetubular cell monolayer was established on the bottom of 8 μm pore sizetranswell inserts and wounded. 3. The inserts are flipped andoxygen-exposed labeled cells are added (2% and 21% exposed cells). Theyare seeded equally at 50,000/cm2. Culturing is in serum-free media at 5%O2 for 24 hrs. 4. Cells that repair wounding are quantified.

FIG. 30B shows that the induction of cells with 2% Oxygen enhanced themigration and wound repair compared to un-induced (21% oxygen). FIG. 30Cplots the % of migrated cells against the migration time. The averagenumber of cells and average percentage of cells are provided in Table16.1.

Hypoxia also induced mRNA expression of CXCR4, MMP9, ICAM1, anddystroglycan; genes that mediate cell migration and attachment. Focalaccumulation of MMP9 and an increase in Connexin 43 aggregates on thecells' plasma membrane was confirmed by immunocytochemistry.

FIG. 31A shows that osteopontin is secreted by tubular cells and isupregulated in response to injury (Osteopontin Immunocytochemistry:Hoechst nuclear stain (blue), Osteopontin (Red), 10×). Osteopontin is asecreted phosphorylated glycoprotein (Kelly et al. J Am Soc Soc Nephrol,1999). Osteopontin is expressed in kidney tubules and is involved inadhesion and migration. Osteopontin is upregulated by injury inestablished tubular cell monolayers as shown by immunoflluorescence(FIG. 31A) and ELISA (FIG. 31B).

TABLE 16.1 3 hr 24 hr Average # Average Average # Average N = 3 cells %cells %  2% O₂ 26.33 61.51% 117.67 60.35% 21% O₂ 16.67 38.49% 76.3339.65% Quantitative image analysis using simple PCI

FIG. 32A shows that the migratory response of cells is mediated in partby osteopontin (Green=migrated cells (5×)). FIG. 32B shows thatneutralizing antibodies (NAb) to osteopontin reduce renal cell migrationresponse by 50%.

FIG. 33 shows that low-oxygen induction of cells modulates expression oftissue remodeling genes. Caveolin 1 is a scaffolding protein involved inmodulation of integrin signaling. MMP9 is a metalloproteinase thatfacilitates migration through extracellular matrix degradation. ICAM1 isan intercellular adhesion molecule associated with epithelial cellmotility. CXCR4 is a chemokine surface receptor that mediates cellmigration.

FIG. 34 depicts a putative mechanism for low oxygen augmentation ofbioactivity of cells leading to renal regeneration.

Taken together, these results suggest that hypoxic exposure facilitatesthe isolation of a specific renal cell subpopulation with demonstratedbioactivity for repair of tubular injury in vitro, and thus maypotentially enhance the ability of these cells to migrate and engraftinto diseased tissue after in vivo delivery. The SRCs demonstrated theability to stabilize renal function and enhance survival in a rodentmodel of progressive CKD. The low oxygen levels (2% O2) provided thefollowing: enhanced post-culture recovery of selected regenerativecells; enhanced cellular attachment and monolayer repair in response totubular injury; and stimulated cellular migration in response to tubularinjury. In addition, cellular migration and attachment were mediated inpart by osteopontin in vitro, low-oxygen upregulated integrins, secretedproteins, and cell adhesion molecules which mediate tissue remodeling,migration, and cell-cell communication.

Example 17—Urine-Derived Microvesicles

An analysis of the miRNAs and proteins contained within the luminalcontents of kidney derived microvesicles shed into the urine wasperformed to determine whether they might be used as biomarkers forassessing regenerative outcome. As excess microvesicles are shed intothe extracellular space, some fuse with neighboring cells while othersare excreted into the urine (Zhou et al. 2008. Kidney Int.74(5):613-621). These urinary microvesicles now become excellentbiomarkers for assay development in order to better understand treatmentoutcomes.

The ZSF1 rodent model of metabolic disease with chronic progressiverenal failure was used. B2+B4 cells were injected into the renalparenchyma of ZSF1 animals. Healthy animals and PBS vehicle were used ascontrols. Urine-derived vesicles were analyzed at different time pointsas summarized below.

-   1: ZSF1 animal—PBS vehicle injected; urine collected 197 days after    injection-   2: ZSF1 animal—PBS vehicle injection; urine collected 253 days after    injection-   3: ZSF1 animal—B2+B4 fraction injected; urine collected 197 days    after injection-   4: ZSF1 animal—B2+B4 fraction injected; urine collected 253 days    after injection-   5. ZSF1 animal—no injection; urine collected on day 197 of the study-   6. ZSF1 animal—no injection; urine collected on day 253 of the study-   7. Healthy animal—no injection; urine collected on day 197 of the    study-   8. Healthy animal—no injection; urine collected on day 253 of the    study

Urine was collected from the test animals on day 197 and about 253 daysafter treatment. Microvesicles were recovered from the urine by standardmethods known in the art (for example, see Zhou et al. Kidney Int. 2008September; 74(5): 613-621). As shown by standard Western blotting inFIG. 35, microvesicles recovered from the urine of treated animals(lanes 3-4) showed an increase in proteins associated with progenitorcells (CD133 & WNT7A) when compared to either vehicle treated (lanes1-2) or untreated controls (lanes 5-8). In fact, microvesicles were onlyrecovered from the urine of diseased animals (lanes 1-6), not healthycontrols (lanes 7-8), as indicated by expression of the microvesiclespecific protein CD63 (FIG. 35). The CD133-containing microvesiclesappear to be prominosomes shed from kidney cells. Both CD133 and WNT7Ahave been associated with regeneration and stem cell division (RomagnaniP and Kalluri R. 2009. Fibrogenesis Tissue Repair. 2(1):3; Lie et al.2005. Nature. 437(7063):1370-5; Willert et al. 2003. Nature.423(6938):448-52; Li et al. 2009. Am J Physiol Renal Physiol.297(6):F1526-33). Taken together, this supports targeting proteinsexpressed in microvesicles as biomarkers for assay development designedto monitor regeneration.

miRNA microarrays and RT-PCR. Microarray and RT-PCR analysis of miRNAfrom urine-derived vesicles was performed by standard methods known inthe art (for example, see Wang et al. supra 2010). In addition toproteins, miRNAs were found within the contents of the isolatedmicrovesicles. Table 17.1 provides examples of miRNAs that were found tobe increased with treatment.

TABLE 17.1 miRNA RQ value miR-15b 6.5206 miR-21 6.4755 miR-30a 6.0002miR-30a* 2.4666 miR-30b-5p 9.8833 miR-30c 6.1688 miR-30d 5.9176 miR-30d*4.1482 miR-30e 8.0836 miR-30e* 2.1622 miR-141 5.1515 miR-146a 2.3054miR-151 3.4462 miR-200a 9.3340 miR-200c 8.0278 miR-429 9.7136

The change in miRNA was analyzed in ZSF1 animals treated with B2+B4 overtime (day 197 and day 253). A fold change was observed for the followingmiRNAs:

miR-370 miR-296 miR-125b-3p miR-362 miR-764 miR-154 miR-1224 miR-221miR-375 miR-22 miR-883 miR-298 miR-598-5p miR-15b miR-671 miR-540miR-143 miR-21 miR-300-3p let-7a let-7e miR-206 miR-339-3p let-7fmiR-222 miR-484 miR-125a-5p miR-433 miR-742 miR-194 let-7b miR-101bmiR-152 let-7c miR-425 miR-128 miR-497 miR-31 miR-423 let-7b* miR-29bmiR-92b miR-24-2* let-7e* miR-139-3p miR-770 miR-345-3p miR-667 miR-124miR-193 miR-98 miR-485 miR-207 miR-181d miR-760-5p miR-501 miR-500miR-103 miR-409-3p miR-493 miR-99b* miR-106b* miR-9* miR-935 let-7dmiR-92a miR-674-5p miR-28* miR-125b-5p miR-743a miR-219-2-3p miR-183miR-352 miR-148b-3p miR-708 miR-326 miR-23b miR-191 miR-181c miR-195miR-181b miR-504 miR-30d miR-186 miR-322* miR-301b miR-872 miR-7amiR-320 miR-188 miR-532-3p miR-652 miR-22* miR-29a miR-322 miR-27amiR-130b miR-429 miR-7b miR-328 miR-291a-5p miR-598-3p miR-345-5pmiR-26a miR-490 miR-29c miR-141 miR-675 miR-215 miR-185 miR-10a-5pmiR-138* miR-27b miR-184 miR-182 miR-200c miR-100 miR-147 miR-431miR-874 miR-664 miR-16 miR-200a miR-339-5p miR-23a miR-30c miR-743bmiR-330* miR-99b miR-96 miR-344-5p miR-212 miR-203 miR-347 let-7imiR-130a miR-125b* miR-30a miR-346 miR-30b-5p miR-199a-5p miR-674-3pmiR-218 miR-25 miR-140* miR-99a miR-99a* miR-192 miR-488 miR-28 miR-30d*miR-342-3p miR-196c miR-151* miR-216a miR-17-3p miR-344-3p miR-101a*miR-133a miR-126* miR-24 miR-19a miR-145 miR-151 miR-211 miR-20b-5pmiR-205 miR-20a miR-294 miR-17-5p miR-34c miR-489 miR-134 miR-871miR-483 miR-296* miR-93 miR-132 miR-26b* miR-9 miR-505 miR-30e*miR-532-5p miR-7a* miR-382 miR-181a let-7i* miR-410 miR-760-3p miR-21*miR-327 miR-140 miR-331 miR-133b miR-29a* miR-434 miR-142-3p miR-138miR-106b miR-324-5p miR-193* miR-30e miR-125a-3p miR-10a-3p miR-350let-7d* miR-877 miR-34a miR-19b miR-29c* miR-219-1-3p miR-223 miR-503miR-543 miR-374 miR-148b-5p miR-210 miR-127 miR-107 miR-30a* miR-26bmiR-34c* miR-365 miR-25* miR-672 miR-378 miR-330 miR-873 miR-196bmiR-146a miR-351 miR-27a* miR-295 miR-292-5p miR-361 miR-196a miR-343miR-10b miR-34b miR-449a miR-466c miR-129 miR-323 miR-29b-2* miR-465miR-541 miR-539 miR-214 miR-761

miRNA levels were analyzed in ZSF1 animals treated with B2+B4 (day 253)and compared to the miRNA levels in ZSF1 animals treated with PBSvehicle (day 253). A fold change was observed for the following miRNAs:

miR-24 miR-186 let-7a miR-195 miR-191 miR-425 miR-871 miR-10b miR-20amiR-30b-5p miR-365 miR-22* miR-19b miR-431 miR-98 miR-99a miR-29c miR-25miR-429 miR-15b miR-194 let-7f miR-21 let-7d miR-200a miR-125b-5pmiR-141 miR-324-5p miR-30c miR-7a* miR-10a-5p miR-30a miR-107miR-148b-3p miR-503 miR-93 miR-100 miR-26a miR-742 miR-16 miR-30dmiR-505 let-7d* miR-743b miR-19a miR-30e miR-9 miR-17-5p miR-200c let-7emiR-743a miR-292-5p miR-23a miR-182 miR-152 miR-322* miR-378 let-7imiR-96 miR-761 miR-351 miR-106b miR-103 miR-434 miR-23b miR-221 miR-26bmiR-27b miR-212 miR-489 miR-29a miR-27a miR-30d* miR-382 miR-29bmiR-148b-5p miR-497 miR-30e* miR-138* miR-184 miR-532-3p miR-465 miR-375miR-199a-5p miR-129 let-7b miR-154 miR-344-3p miR-133a miR-127 miR-330miR-345-5p miR-667 miR-374 miR-342-3p miR-92a miR-10a-3p miR-34amiR-140* miR-352 miR-7a miR-322 miR-181a miR-291a-5p miR-181b let-7i*miR-34c miR-296* miR-134 miR-300-3p miR-339-3p miR-196a miR-219-1-3pmiR-28* miR-203 miR-30a* miR-24-2* let-7c miR-125a-5p miR-345-3p miR-151miR-323 miR-330* miR-20b-5p miR-327 miR-504 miR-26b* miR-883 miR-449amiR-140 miR-541 miR-210 miR-128 miR-708 miR-188 miR-222 miR-196c miR-410miR-29a* miR-216a miR-101a* miR-872 miR-146a miR-147 miR-328 miR-27a*miR-192 miR-760-3p miR-223 miR-296 miR-295 miR-106b* miR-196b miR-151*miR-466c miR-433 miR-138 miR-490 miR-17-3p miR-211 miR-326 miR-350miR-339-5p miR-664 miR-142-3p miR-423 miR-770 miR-23a* miR-101b miR-652miR-674-5p miR-219-2-3p miR-183 miR-214 let-7e* miR-22 miR-540 miR-484miR-485 miR-133b miR-1224 miR-935 miR-29c* miR-320 miR-532-5p miR-758miR-675 miR-764 miR-362 miR-105 miR-193 miR-34b miR-874 miR-672miR-409-3p miR-500 miR-325-5p miR-185 miR-501 miR-760-5p miR-344-5pmiR-347 miR-346 miR-294 miR-338 miR-301a miR-31 miR-671 miR-488miR-125b-3p miR-34c* miR-539 miR-204* miR-92b miR-132 miR-361 miR-29b-2*miR-126* miR-711 miR-298 miR-99b miR-215 miR-139-3p miR-125a-3p miR-301bmiR-342-5p miR-181d miR-206 miR-877 miR-543 miR-370 miR-99a* let-7b*miR-130a miR-129* miR-99b* miR-193* miR-181c miR-463 miR-873 miR-21*miR-146b miR-218 miR-25* miR-343 miR-124 miR-143 miR-205 miR-130b

miRNA levels were analyzed in ZSF1 animals treated with B2+B4 (day 197)and compared to the miRNA levels in ZSF1 animals treated with PBSvehicle (day 197). A fold change was observed for the following miRNAs:

miR-143 miR-24-2* miR-98 miR-370 miR-26b miR-434 miR-351 miR-375miR-339-5p let-7a let-7f miR-296 miR-152 miR-206 miR-667 miR-141 miR-29amiR-181b let-7c miR-100 miR-324-5p miR-222 miR-29c miR-30e miR-362miR-16 miR-10a-5p miR-200a miR-96 miR-125a-5p miR-188 miR-151 miR-29bmiR-429 miR-125a-3p miR-28* miR-505 miR-195 miR-106b* miR-21 miR-210miR-30a let-7e miR-742 miR-423 miR-182 miR-30d miR-19b let-7b miR-194miR-500 let-7i miR-433 miR-92a miR-200c miR-23b miR-291a-5p miR-99amiR-124 miR-181d miR-221 miR-101b miR-320 miR-30b-5p miR-497 miR-345-3plet-7d miR-425 miR-764 miR-103 miR-347 miR-191 miR-148b-3p miR-19amiR-10b miR-26a miR-431 miR-298 miR-186 miR-17-5p miR-92b miR-22 miR-374miR-203 miR-330* miR-664 miR-130b miR-484 miR-877 miR-449a miR-339-3pmiR-24 miR-7a* miR-106b miR-205 miR-219-2-3p miR-25 miR-196b miR-23 amiR-326 miR-126* miR-322 miR-129 miR-20b-5p miR-181a miR-31 miR-128miR-219-1-3p miR-34a miR-22* miR-30d* miR-652 miR-196c miR-301b miR-15bmiR-192 let-7e* miR-130a miR-151* miR-196a miR-378 miR-134 miR-9 miR-30cmiR-214 miR-27a* let-7d* miR-674-5p miR-488 miR-874 miR-125b-5p miR-183miR-485 miR-365 miR-26b* miR-93 miR-532-3p miR-138 miR-671 miR-29c*miR-382 miR-99b* miR-7a miR-760-3p miR-139-3p miR-147 let-7i* miR-27bmiR-27a miR-184 miR-21* miR-181c miR-25* miR-328 miR-99b miR-34c miR-185miR-125b-3p miR-30a* miR-743b miR-193 miR-466c miR-127 miR-342-3p miR-28miR-345-5p miR-215 miR-142-3p miR-140* miR-132 miR-107 miR-20amiR-532-5p miR-148b-5p miR-331 miR-17-3p miR-483 miR-218 miR-7b miR-223miR-30e* miR-34b miR-361 miR-330 miR-193* miR-503 miR-344-5p miR-216amiR-873 miR-493 miR-99a*

The miRNAs listed in Table 17.1 provide examples of miRNAs that havebeen implicated in processes relative to tissue regeneration. miR-15bhas been implicated in regulating apoptosis through BCL-2 and caspaseregulation (Guo et al. 2009. J Hepatol. 50(4):766-78) as well as cellcycle progression through the regulation of cyclins (Xia et al. 2009.Biochem Biophys Res Commun. 380(2):205-10). miR-21 was shown to inhibitapoptosis by modulating survival pathways MAPK/ERK. The miR-30 family ofmiRNAs is critical for podocyte structure and function suggesting thatan increase maybe necessary for glomerulargenisis. miR-141, 200a, 200cand 429 are all involved in modulating epithelial to mesenchymaltransition (EMT) in response to TGF-β signaling possibly reducingfibrosis (Saal et al. 2009. Curr. Opin. Nephrol. Hypertens. 18:317-323).miR-146a and 151 have been implicated in NFκB modulation thuspotentially reducing the inflammatory response in vivo (

Taganov et al. 2006. Proc Natl Acad Sci USA. 103(33):12481-6;Griffiths-Jones et al. 2006. NAR. 34 Database Issue: D140-D144).Collectively, these miRNAs regulate processes related to a successfulregenerative outcome; thus making them candidate biomarkers for assaydevelopment. Overall, this data supports the concept that urinarymicrovesicles and/or their luminal contents are viable targets forregenerative assays as they contain proteins and miRNAs capable ofmodulating multiple pathways including: TGF β-1, NFκB, apoptosis, celldivision and pluripotency in addition to providing practitioners with anon-invasive means of monitoring treatment.

1-34. (canceled)
 35. A method of assessing whether a kidney disease (KD)patient is responsive to treatment with a therapeutic, the methodcomprising detecting the amount of vesicles or their luminal contents ina test urine sample obtained from a KD patient treated with thetherapeutic, as compared to or relative to the amount of vesicles ortheir luminal contents in a control urine sample, wherein a higher orlower amount of vesicles or their luminal contents in the test urinesample as compared to the amount of vesicles or their luminal contentsin the control urine sample is indicative of the patient'sresponsiveness to treatment with the therapeutic.
 36. The method ofclaim 35, wherein the therapeutic comprises an enriched population ofrenal cells.
 37. The method of claim 36, wherein the enriched populationof renal cells is enriched for renal tubular cells.
 38. The method ofclaim 37, where the enriched population of renal cell cells furthercomprises epithelial cells of the collecting duct system.
 39. The methodof claim 35, wherein the vesicles comprise a biomarker.
 40. The methodof claim 39, wherein the biomarker is miRNA.
 41. The method of claim 40,wherein the miRNA comprises one or more of miR-370, miR-362, miR-1224,miR-22, miR-598-5p, miR-540, miR-300-3p, miR-206, miR-222, miR-433,let-7b, let-7c, miR-497, let-7b*, miR-24-2*, miR-770, miR-124, miR-485,miR-760-5p, miR-296, miR-764, miR-221, miR-883, miR-15b, miR-143,let-7a, miR-339-3p, miR-484, miR-742, miR-101b, miR-425, miR-31,miR-29b, let-7e*, miR-345-3p, miR-193, miR-207, miR-501, miR-125b-3p,miR-154, miR-375, miR-298, miR-671, miR-21, let-7e, let-7f, miR-125a-5p,miR-194, miR-152, miR-128, miR-423, miR-92b, miR-139-3p, miR-667,miR-98, miR-181d, miR-500, miR-103, miR-99b*, miR-935, miR-674-5p,miR-743a, miR-352, miR-326, miR-181c, miR-504, miR-322*, miR-7a,miR-532-3p, miR-29a, miR-130b, miR-328, miR-345-5p, miR-29c, miR-215,miR-138*, miR-182, miR-147, miR-664, miR-339-5p, miR-743b, miR-96,miR-203, miR-130a, miR-346, miR-674-3p, miR-140*, miR-192, miR-409-3p,miR-106b*, let-7d, miR-28*, miR-219-2-3p, miR-148b-3p, miR-23b, miR-195,miR-30d, miR-301b, miR-320, miR-652, miR-322, miR-429, miR-291a-5p,miR-26a, miR-141, miR-185, miR-27b, miR-200c, miR-431, miR-16, miR-23a,miR-330*, miR-344-5p, miR-347, miR-125b*, miR-30b-5p, miR-218, miR-99a,miR-488, miR-493, miR-9*, miR-92a, miR-125b-5p, miR-183, miR-708,miR-191, miR-181b, miR-186, miR-872, miR-188, miR-22*, miR-27a, miR-7b,miR-598-3p, miR-490, miR-675, miR-10a-5p, miR-184, miR-100, miR-874,miR-200a, miR-30c, miR-99b, miR-212, let-7i, miR-30a, miR-199a-5p,miR-25, miR-99a*, miR-28, miR-30d*, miR-151*, miR-344-3p, miR-126*,miR-145, miR-20b-5p, miR-294, miR-489, miR-483, miR-132, miR-505,miR-7a*, let-7i*, miR-21*, miR-331, miR-434, miR-106b, miR-30e, miR-350,miR-34a, miR-219-1-3p, miR-543, miR-210, miR-30a*, miR-365, miR-378,miR-196b, miR-27a*, miR-361, miR-10b, miR-466c, miR-342-3p, miR-216a,miR-101a*, miR-24, miR-151, miR-205, miR-17-5p, miR-134, miR-296*,miR-26b*, miR-30e*, miR-382, miR-410, miR-327, miR-133b, miR-142-3p,miR-324-5p, miR-125a-3p, let-7d*, miR-19b, miR-223, miR-374, miR-127,miR-26b, miR-25*, miR-330, miR-146a, miR-295, miR-196a, miR-34b,miR-129, miR-196c, miR-17-3p, miR-133a, miR-19a, miR-211, miR-20a,miR-34c, miR-871, miR-93, miR-9, miR-532-5p, miR-181a, miR-760-3p,miR-140, miR-29a*, miR-138, miR-193*, miR-10a-3p, miR-877, miR-29c*,miR-503, miR-148b-5p, miR-107, miR-34c*, miR-672, miR-873, miR-351,miR-292-5p, miR-343, miR-449a, miR-323, miR-29b-2*, miR-539, miR-465,miR-214, miR-541, miR-761, miR-105, miR-338, miR-342-5p, miR-129*,miR-325-5p, miR-301a, miR-463, miR-146b, miR-23a*, miR-758, miR-204*,miR-711 and miR-378.
 42. The method of claim 40, wherein the miRNAcomprises one or more of miR-222, miR-30b-5p, miR-449a, miR-146a,miR-130a, miR-23b, miR-21, miR-124, miR-151, miR-23a, miR-30c, miR-1224,miR-92a, miR-100, miR-125b-5p, miR-195, miR-10a-5p, miR-15b, miR-30a*,miR-30d, miR-30e*, miR-429, miR-30d*, miR-141, miR-200a, miR-30a,miR-30e, miR-200c, miR-24, miR-195, miR-871, miR-19b, miR-99a, miR-429,let-7f, or miR-324-5p.
 43. The method of claim 42, wherein the miRNAcomprises miR-222.
 44. The method of claim 40, wherein the miRNAcomprises one or more of miR-30b-5p, miR-449a, miR-146a, miR-130a,miR-23b, miR-21, miR-124, or miR-151.
 45. The method of claim 40,wherein the miRNA comprises one or more of miR-21, miR-23a, miR-30c,miR-1224, miR-23b, miR-92a, miR-100, miR-125b-5p, miR-195, ormiR-10a-5p.
 46. The method of claim 40, wherein the miRNA comprises oneor more of miR-146a, miR-130a, or miR-23b.
 47. The method of claim 40,wherein the miRNA comprises one or more of miR-15b, miR-30a*, miR-30d,miR-30e*, miR-151, miR-429, miR-21, miR-30b-5p, miR-30d*, miR-141,miR-200a, miR-30a, miR-30c, miR-30e, miR-146a, or miR-200c.
 48. Themethod of claim 40, wherein the miRNA comprises one or more of miR-24,miR-195, miR-871, miR-30b-5p, miR-19b, miR-99a, miR-429, let-7f,miR-200a, miR-324-5p, or miR-10a-5p.
 49. The method of claim 39, whereinthe biomarker comprises a protein.
 50. The method of claim 49, whereinthe protein comprises CD133.
 51. The method of claim 49, wherein theprotein comprises WNT7A.
 52. The method of claim 35, wherein thevesicles comprise microvesicles.